Production of Secondary Metabolites by Solid-State Fermentation

01996 Elsevier Science B.V. All rights reserved. Biotechnology Annual Review Volume 2. M.R. El-Gewely, editor.

85

Production of secondary metabolites by solid-state fermentation Javier Barrios-GonzBlez and Armando Mejia Departmento de Biotecnologia, Universidad Autbnoma Metropolitana-lztapalapa.Apdo., Mexico

Abstract. Microbial secondary metabolites are useful high value products that are normally produced by liquid culture; but could be advantageously produced by solid-state fermentation (SSF). Particularly if SSF could benefit from a deeper understanding of microbial physiology in a solid environment. Recent research indicates that different kind of secondary metabolites can be produced by SSF: antibiotics, phytohormones, food grade pigments, alkaloids, etc. Physiology in SSF shows several similarities with physiology in liquid medium, so similar strategies must be adapted for efficient processes. However, there are certain particularities of idiophase in solid medium which dictate the need for special strains.

Key words: alkaloids, antibiotics, basic principles, control of metabolism, effect of environmental and nutritional factors, key variables, phytohormones, pigments, production, secondary metabolites, solid-state fermentation (SSF), strain improvement.

Introduction Solid-state fermentation (SSF) is an ancient microbial culture system that is being transformed for new purposes, using new approaches of microbiology, biochemistry and biochemical engineering. There are advantages in employing SSF processes, at least for certain applications, over the conventional submerged fermentation (SmF) ones. However, the latter is usually chosen, owing to the great success of large-scale commercial installations of this kind in many fields of biotechnology. Such units are excellent examples of how fundamental microbiological and biochemical knowledge has been applied to bioengineering principles which have set the procedures to design, control and optimize them. Most of the processes using the SSF technique are commercialized in oriental countries, mainly in Japan. Nevertheless, a resurgence of interest has occurred in Western countries over the last 1 e 1 5 years [1,2] in response to the ever rising demand for economy in processes. This is a well-justified trend which ultimately may lead to an extensive industrialization of SSF throughout the world [3]. Mitchell and Lonsane [3] have divided SSF processes into socioeconomic and profit-economic applications. Socioeconomic applications are either small scale processes performed to contribute something essential for the community (like ensiling of grasses and upgrading lignocellulosic products or staple foods) or involve the disposal of wastes (e.g., composting). A profit-driven application on the other

Address for correspondence: Javier Barrios-Gonzilez, Departmento de Biotecnologia, Universidad Aut6noma Metropolitana-Iztapalapa. Apdo. Postal 55-535, 09340 Mexico.

86 hand will seek commercial profit. In the short term, new profit-economic SSF processes are most likely to come from Japan, which is not only highly industrialized but also has significant experience with large scale SSF because of koji industry. In the Western society traditional applications of SSF are very scarce and SSF has been largely neglected since the Second World War. Soy sauce production, which involves the SSF step of koji fermentation, is an example of an originally small-scale traditional process which has become highly industrialized [3]. The advancement of SSF technology has permitted a higher degree of control over the process and hence, the production of high value products like antibiotics and other secondary metabolites. These compounds are characterized by the great variety of biological activities that confer their actual or potential use in different industrial fields e.g., pharmacy, agriculture and livestock. Normally these sophisticated metabolites would be produced by SmF, but the research performed in the last 10 years indicates that these compounds could be produced in great amounts, and probably with advantages, by SSF. Secondary metabolites are generally reported to be produced in higher concentrations in solid culture, often in shorter times and without the need of aseptic conditions [4-81. Capital costs are claimed to be significantly less [9]. In some instances, economic performance could be even better if the final product is required in solid form, for example antibiotics in animal feeds. However, if the use of SSF is going to generalize, several problems have to be solved. A serious problem with- SSF is the inexperience of engineers in Western nations in the design and scale-up of solid-state fermenters; and the nonexistence of standard fermenters for these processes.- Nevertheless, there have been noticeable advances in this field, which have been reviewed by several authors [lo-121. A very important disadvantage of SSF is the relatively little data on the physiology and genetics of SSF strains in solid medium, to enable optimization of production. In consequence SSF processes can be underestimated or their full potential not exploited. The aim of this work is to analyze the literature on production of secondary metabolites in SSF, and to start drawing conclusions about fundamental principles of microbial secondary metabolism in solid medium. As in the case of SmF, this basic principle will eventually be the basis for efficient process development, control strategies and reactor design, as well as for the methodology to generate hyperoducing strains, particularly suited for solid conditions. The first two sections describe general features of secondary metabolism and SSF. Later sections discuss the control of secondary metabolism in solid medium by environmental and nutritional factors (including the role of regulatory mechanisms), while the last section deals with the relation between the strain and the production level obtained in SSF.

Secondary metabolites The early microbial physiologists observed that in the logarithmic phase of growth

87 there is an intense metabolism, with microbes replicating rapidly their cellular components, as a prerequisite for growth and cell division. It was assumed initially, that stationary phase represented a complete metabolic inactivity of the microorganisms. It was the chemists of natural products who realized the falsehood of that concept. Between 1920 and 1930, organic chemists found that fungal cultures in stationary phase were an almost inexhaustible source of complex organic compounds. As the structures of these molecules were described, it became evident that these compounds did not play a role during exponential phase of growth. Some years before, plant physiologists had recognized two similar classes of compounds produced by plants. There were compounds like chlorophylls that were synthesized by all plants; these were named primary products of metabolism. Conversely, there were compounds like camphor and tannins (or carbinol), which were only produced by particular species of plants, and no metabolic function could be assigned to them. These were called secondary products of metabolism. A little more than 2 decades ago the term was used to describe a wide range of compounds produced by microorganisms, and as their plant counterparts, are not directly related to compounds that form the cell. Today the term secondary metabolite is more often associated to microbial products than plant products [13]. There is still a certain amount of controversy about the lack of function of secondary metabolites, since in some cases specific functions have been attributed to individual secondary metabolites, (mostly related to processes of differentiation) or, in other cases, an antagonistic role in natural environment has been proposed [14]. Hence an adequate definition is the one proposed by Demain et al. [ 151: metabolites that are not essential for vegetative growth of the producing organism in pure culture. They are usually formed as a mixture of closely related members of a chemical family. For example, there are at least eight aflatoxins, 10 polimixins over 20 penicillins and 20 actinomycines. Each is produced by a narrow taxonomic range of organisms and production ability is easily lost by mutation (strain degeneration). These compounds have been described as secondary metabolites in opposition to primary metabolites like amino acids, nucleotides, lipids and carbohydrates that are essential for growth. Despite the enormous diversity of chemical structures found in microbial secondary metabolites, most of these compounds can be grouped in a few classes depending on their biosynthetic origin. A convenient classification is the one proposed by Rose [13]. This author classifies these metabolites in only four groups or families, and enphasizes that this reflects the reduced number of groups in which low molecular weight compounds, precursor to of the cell constituents, can be classified. In this way, secondary metabolites derive from: a) amino acids; b) sugars; c) acetyl-CoA (and related compounds, including Krebs cycle intermediates); and d) terpenes.

Growth and secondary metabolism The relationship between growth and secondary metabolisms has been extensively studied [16-18]. It has been found that production of secondary metabolites starts when growth is limited by the exhaustion of one (or more) key nutrient. Bu’Lock introduced the term trophophase to name the phase of primary metabolism; and idiophase for secondary metabolism. Although these terms have a descriptive value, they are not absolute or mutually exclusive. It is considered that growth in the nonlimited phase (trophophase) is balanced and all the biosynthetic capacity of the cell is required to cope with a high growth rate. Growth is unbalanced when growth is limited by the shortage of a nutrient. Those cell processes that require the limiting nutrient are restricted, while those which do not require it are not. Since growth depends of a wide range of biosynthetic activities, the absence of one (or several nutrients) e.g., carbon, nitrogen, phosphate or trace elements, can restrict it. Not limiting nutrients can then be diverted towards biosynthesis not related to growth. Biosynthesis of secondary metabolites or specific compounds required for differentiation can be stimulated in this way. Consequently, a popular hypothesis proposes that the specific products of secondary metabolism are not important but the process of secondary metabolism itself is of selective advantage for the microorganism. It is considered to provide a mechanism by which excess intermediates can be metabolized in an adverse environment. Such a mechanism would serve to maintain the cell in a functional state during conditions which prevented growth [ 191. It is not possible to define a specific growth rate below which growth can be said to be of the idiophase type. However, such a growth rate (p) can be defined for a specific metabolite produced in the idiophase [20]. These ideas can be illustrated by mentioning the example of ergot alkaloids whose synthesis starts when phosphate source is exhausted from the medium. In gibberellic acid fermentation the growth rate is controlled by limiting the supply of nitrogen. Chemostat studies using glycine as the nitrogen source have shown that pmaxfor Gibberella fujikuroi is approximately 0.18/h and at growth rates approaching this value growth is balanced. At growth rates of O.lO/h carbohydrates accumulated but no bikaverin or gibberellic acid synthesis occurred. Growth rates of less than O.O5/h were required for bikaverin formation and of less than O.Ol/h for gibberellic acid synthesis [21]. In the case of penicillin, whose synthesis correlates with the exhaustion of the easily metabolized carbon source (glucose), lactose is used to reduce the growth rate of P. chrysogenum and stimulate penicillin biosynthesis. The molecular events that turn on secondary product formation at the end of the trophophase are unknown. It is considered that nutrients limitation cause an inducer (positive effector) of secondary metabolic enzymes to accumulate or release genes of secondary metabolism from catabolic repression (negative effector). In any case it is well known that at the end of the trophophase enzymes specifically related to formation of secondary products suddenly appear [22]. It has been demonstrated that the production of secondary metabolites is affected by regulatory mechanisms that appear to be similar to the ones that control primary metabolism, i.e., induction,

89 feedback regulation and catabolite regulation [ 2 3 ] . In this way, production of many secondary metabolites is negatively affected by a number of growth medium constituents, falling into three broad classes: carbon/energy sources, nitrogen sources, and phosphorus-containing compounds. Commercial importance Figure 1 shows the interrelation between the products of secondary metabolism and primary metabolism. It also gives an idea of the great variety of compounds produced. A consequence of the wide range of chemical structures of secondary metabolites is the enormous range of biological activities that can be found among these compounds. Hence, many secondary metabolites have great economic importance since they are applied to or have potential applications in different fields. This can be illustrated by the following examples: 1. Pharmaceutical industry: antibiotics, antitumorals, inmunodepressors, etc. 2. Agriculture: plant growth stimulants, antibiotics with herbicide and fungicide activities that would be more environmental friendly. 3. Cattle raising: growth promoters (antibiotics), antihelmints and coccidiostatics. 4. Food industry: food grade colorants and aromas. These compounds are or would normally be produced in liquid culture (submerged fermentation). However, at least some of them could be advantageously produced by SSF.

SSF SSF has been used since antiquity for the preparation of fermented foods, silages and composting. The use of koji for soy sauce in China, Japan and Southeast Asia goes back as far as 1,000 years BC, and can be considered as a prototype of SSF. It consists in the cultivation of Aspergillus oryzae on soybeans and other grains to produce proteases and amylases, which degrade proteins and transform starch into sugars. In this way, the fermented material is used for the production of soy sauce or, in a second stage, rice wine or sak6. Although the earliest commercial production of enzymes depended on SSF technology developed in China and Japan, the advent of sterile submerged culture techniques in the 40s displaced the solid-state methods in the Western countries [24]. Work performed by Hesseltine et al. [8,25,26] studied and described the traditional SSF processes of the East, and informed about the great technological importance of these culture systems. These reports are probably responsible, in great part, for the renewed interest in SSF observed during the last 10 or 15 years. As a consequence, SSF is being transformed for new purposes, using new approaches of microbiology, biochemistry and biochemical engineering, and often presents several advantages over SmF.

90 Centanycine

Kanvnycins DNA, RNA

phaphalr

c H-

1

Triov phorphate Serine

t

- -+

Pe&

phosphate--

Tetfose phorphate

Candiddin Chinramphenid

Shikimate

1

RrimFin

Phosphadmate

,

-

hidonate IMpIonyI CON

1

Fatty acas

Fig. 1. Relation between primary and secondary metabolism (modified from Rehacek and Sajdl, 1990) [102].

Modern SSF systems Besides the koji-type systems, SSF cultures are now performed on other starchy substrates like roots (cassava or potato flour), bananas, etc. or lignocellulosic material like straw or wood pulp. These SSF systems are referred to as nontraditional in this review. A new type of SSF uses an inert support with absorbed liquid medium. The support can be of natural origin like sugarcane bagasse, or artificial (or synthetic) like polyurethane, amberlite or vermiculite. This kind of SSF is very useful for basic studies since liquid media, with the desired composition can be used, and fermented broths can be extracted And analyzed at any time. Some of them, like sugarcane bagasse, also show great productivity (Fig. 2) [4,27,28]. SSF was defined by Hesseltine [25] as a fermentation in which the substrate is not liquid. However, a modem definition is the one proposed by Lonsane et al. [9], as a microbial culture that develops on the surface and at the interior of a solid matrix and in absence of free water. The porous matrix can be a moistened substrate or an inert support capable of absorbing nutrients dissolved in a solution. In this way two types of SSF can be distinguished, depending on the nature of the solid phase used:

91

Fig. 2. Laboratory scale column reactor packed with 12 g of sugarcane pith bagasse impregnated with liquid medium.

a) Solid culture of one support-substrate phase. Solid phase is constituted by a

material that assumes, simultaneously, the functions of support and of nutrients

Fig. 3. Pilot bioreactor for gibberellic acid production by SSF. This reactor works under sterile conditions and has a highly sophisticated control system (Solar et al. Depto. de Ing. Quimica y de Bioprocesos, Pontificia Universidad Cat6lica de Chile, Chile).

92

source. This material is generally of starchy or lignocellulosic nature. Most of the applications of SSF use this kind of system. b) SSF of two substrate-support phases. Solid culture with an inert support impregnated with a liquid medium. -In this type of fermentation, solid phase is considered as an inert support that serves as a reservoir for a nutritive solution. In this type of SSF water-holding capacity is of paramount importance in the selection of the support. New applications

SSF processes are used on a commercial scale for the production of different types of traditional fermented foods, fungal metabolites and for bioconversion of organic wastes in the East, mainly in Japan [3,29]. However, this culture system could have significative advantages over SmF methods for the manufacture of nontraditional products of interest. These advantages and the versatility of this culture system has driven the appearance of a great number of new application fields (Table 1) [29]. Table 1 . Examples of new applications of SSF. Application field

Product

Microorganisms

Ref.

Fermented foods

Cheeses Koji Pozol Bread Cacao

Penicillum spp. Aspergillus orizae Lactobacillus spp. and yeasts Yeasts and Acetobacter

[301 [311 [321 [331 1331

Protein enrichment of food stuff

Lignocellulosic fibers Starchy material

Aspergillus terreus Aspergillus niger

[341 [311

Enzymes production

Amylases Proteases Cellulases Pectinases

Aspergillus Aspergillus Trichoderma Aspergillus

[351 1361 [371 1381

Primary metabolites

Citric acid Galic acid Gluconic acid

A. niger A. niger A. niger

Secondary metabolites

Mycotoxins

A. flaws and other fungal species

181

Alcohol production

Ethanol

Saccharomyces spp.

[401

Spore production

Inoculum Biological control

Penicillium spp. Trichoderma hartzianum

[411 1371

Composting

Compost

Mixed flora

1421

Silage

Silage

Lactobacillus spp.

[431

Edible mushrooms

Pleurotus

Pleurotus spp.

[441

Biological filters

Treated water

Mixed flora

[421

93 Advantages and disadvantages SSF and SmF have been compared by several authors [9,12,26,45]. Advantages of SSF include: 1. Often sterile conditions are not required. The process proceeds as a pure culture since solid conditions (similar to their natural habitat) ecologically favor fungi and actinomycetes. 2. Mycelial morphology of fungi and actinomycetes (microorganisms most associated with secondary metabolite formation) is well suited to invasive growth on solid medium. This morphology is responsible for considerable difficulties in large-scale submerged culture. These include highly viscous, non-Newtonian broths and foam production. This results in very high power requirements for mixing and oxygen transfer efficiency and can lead to problems during product recovery ~461. 3. Conversely, energy requirements in SSF are relatively low since oxygen is transferred directly to the microorganisms. 4. Metabolites are often produced in much higher yields [4-81. This is an important point to counterbalance disadvantages described below. 5 . Quite often, the solid-state fermented product is used as such, requiring little downstream processing and, hence, improving the process economics relative to liquid-culture-based processes. In some instances the final product is required in solid form, e.g., antibiotics in animal feeds. 6. Extraction of soluble products from solid-state mash into a small volume of solvent can often yield a concentrated solution of the product, which once again, is amenable to purification by simpler means. This implies a reduction in liquid effluents. 7. Capital costs are claimed to be significantly less [9]. 8. Very concentrated media can be used without a deleterious effect on product formation. 9. Useful changes in enzyme characteristics when produced in SSF. Regarding secondary metabolites, secretion of pigments (intracellular in SmF) into the solid medium has been reported (see section on: Moisture content and water activity). Notwithstanding the advantages, large scale commercial use SSF requires solutions to several problems. Disadvantages of SSF include: 1. Heat dissipation problems. 2. Lack of sensors and efficient methods to handle solids. 3. Difficult to add nutrients and controlling agents. 4. The inexperience of engineers in Western nations in the design and scale-up of solid-state fermenters; and the nonexistence of standard fermenters for these processes. 5. A very important disadvantage is the relatively little data on the physiology and genetics of SSF strains to enable optimization of production.

94 Physiological aspects The physiological effect of the liquid environment on biomass and product formation by filamentous fungi has been extensively studied in submerged culture [20,48]. However, there is little information available of the effect of the solid environment on microorganisms in SSF. It can be thought that the effects are similar to the ones observed in liquid cultures, modified for the chemical composition, physical structure of the substrates and local variations of temperature, pH and nutrients concentration and dissolved gases [ 121. Nevertheless, only recently did it become apparent that the biochemical and physiological response of certain microorganisms in solid-state culture may differ greatly from those in SmF, leading to reduction in, or even total loss of, productivity of the desired enzyme or metabolite. Physiology in solid medium can be so different that microorganisms produce enzymes with different characteristics of size, Km, stability, and optimum pHs and temperatures, and a different sensitivity to substrate inhibition in SSF [49,50]. It has even been reported that intracellular enzymes become extra cellular when produced in SSF [51,52]. Process variables A key question for the industrial application of many of these processes is: what are the key process variables in SSF that control growth and microbial metabolism? In practice SSFs are controlled with the following process variables [12,53]: a) pretreatment; b) nutrients; c) particle size; d) moisture content; e) sterilization; f) temperature and pH; g) aeration and agitation; and h) inoculum size; In fact these are the same parameters that are controlled in SmF (except moisture content and particle size). This does not mean that the effect of these parameters is well understood in SSF, rather, that the concepts of SmF are applied directly (Fig. 3). Some theoretical considerations on the effect of these variables on microbial growth in SSF are commented below. 1. Pretreatment. Natural solid substrates generally require some kind of pretreatment to make their chemical constituents more accessible and their physical structure more susceptible to mycelial penetration and adhesion. 2. Nutrients. Although most traditional food fermentations do not require nutritional supplementation, it may be beneficial in nontraditional fermentations. Nutritional factors are usually limiting for microbial growth [54]. In solid materials this limitation is more severe due to the limited diffusion rates and the limited access of the fungus to it.

95

3.

4.

5. 6.

In fact the understanding of this subject is very poor, probably because most of the processes used are of one support-substrate phase. In this way the medium is already adequate for growth or is balanced with nitrogen and phosphorous sources. Particle size. It is generally considered that smaller sizes provide greater superficial area for transference of heat and gas exchange. It also results in higher superficial concentrations of nutrients and shorter pathways for their diffusion [12]. A smaller particle size results in a higher packing density and a concomitant reduction of the void space between particles. This tends to reduce the area of transference of heat and gas exchange. Moisture content. Water is a main component of microorganisms and in SSF has an important role in enzymes, nutrients and products diffusion through the solid matrix [35].It is noteworthy that free water must not be too abundant since it may reduce porosity and, in consequence, decrease the gases exchange. On the other hand, variations in moisture content are produced during the course of the fermentation. The cause of these changes are evaporation, water production by respiration of the culture and, possibly, moisture exchange between the air and the solid medium. Due to the importance of moisture content and water activity of the solid medium on microbial physiology, several authors have performed studies on this parameter. Oriol et al. [55] did interesting estimations of total water, consumed water and residual water in a SSF on cassava flour (one supportsubstrate phase). A theoretical calculation, based on the Ross equation, indicated that water activity of the solid medium descended to 0.85 at the end of the culture. The authors interpreted this as the cause of growth cessation. It is important to note that this theoretical Aw is different from the actual measurement, which is a mixture of Aw of the solid substrate and the Aw of the mycelium in it. Sterilization. Many SSF exclude or greatly reduce the problem of bacterial contamination, so many processes require no sterilization and/or nonsterile conditions during the process. Temperature and pH. In SSF metabolic heat generation can provoke an increase in temperature in the reactor and cause a serious problem [45]. This is due to the high local substrate concentration, the low water content and weak thermal conductivity of the biological materials [53,56,57]. With the aim of solving this problem several authors have established strategies for temperature regulation. Most of them use forced convection of air through the fermenter. Others have used the cooling properties of water evaporation to automatically control moisture and temperature [58-601. We consider that the solution involves the use of all these strategies plus water addition to compensate losses from evaporative cooling. Global pH of the liquid phase of a SSF can be considerably different than the local pH values on the solid surfaces where growth is taking place, due to the superficial charge effects and ionic equilibrium modified by the effect of solute

96 transport [12]. Although there is not a standard protocol for pH measurement, a general procedure is to determine global pH, after suspension of a sample in a 10 times greater volume of water. 7 . Aeration. Aeration fulfills four main functions in SSF, namely 1) maintain aerobic conditions; 2) desorption of CO,; 3) regulate temperature and 4) regulate the moisture level [61]. Partial pressures of CO, and 0, of the gaseous atmosphere of a SSF are important factors for growth and product formation. Oxygen should be enough not to limit growth, and is controlled by the air flow in the reactor. 8. Znoculum size. Generally, SSFs are inoculated with high spore concentrations. Mudgett [ 121 indicates that it is necessary to optimize this parameter, since a very low inoculum density can give insufficient biomass and allow growth of contaminants. Saucedo-Castafieda [62] studied the effect of inoculum size (5.7 x lo7 to 3.6 x lo3 cells/ml) on growth of yeast on SSF on sugarcane bagasse. Shorter lag phases were obtained with increased inoculum sizes. Specific growth rates of the culture also increased at increased inoculum sizes (it stabilized in 5.8 x lo5), while final biomass concentration seemed less sensitive to this factor. Experiments performed by Oriol et al. [63], with Aspergillus niger in the same SSF system, confirm these results (shorter lag phases), using inocula of 5.8 x lo5 to lo9 spores/g of dry medium. It is interesting to note that an inhibitory effect on germination has been observed at those spore densities in liquid culture [64]. Mycotoxin production by SSF Mycotoxins are dangerous metabolites of fungal origin, some of which can represent a health hazard in very small quantities. Although mycotoxins are secondary metabolites, its production in SSF is discussed in this previous section for historical reasons. The fact that mycotoxins are harmful metabolites is another reason to separate them from the useful high-valued compounds. In any case, knowledge of secondary metabolism in solid medium can also be applied to avoid mycotoxin contamination of stored agricultural goods, or even SSFs [65,66]. Research on SSF in the Western countries practically started with the studies on mycotoxin production. Hesseltine and co-workers, in the Northern Regional Research Center (Department of Agriculture of the USA) in Peoria, Illinois, adapted the koji SSF system to produce mycotoxins in much higher concentrations than the ones that could be obtained by liquid fermentation [8,26]. The authors explain that production of great amounts of aflatoxins was entrusted to them by the government for field trails. Initially they tried production by liquid fermentation with very unsatisfactory yields. After that they applied the methodology of rice koji with much higher yields. A very surprising increase in production came when a flask with the rice fermentation was agitated in shaker (200 rpm) as if it contained liquid medium. Very impressive concentrations of aflatoxins were obtained in this way (1.5 g/kg). The method was

97

adopted by the group to produce mycotoxins from different species with excellent results. Similar concentrations of ochratoxins were obtained with some species of Aspergillus cultivated on wheat (production was lower on corn) [25]. Ochratoxin was produced in concentrations up to 2.4 g/kg on wheat, by means of a rotating drum fermenter of four compartments, with deflectors that rise and drop the grains in each revolution [67]. The highest rotating speed tested (16 rpm) gave the highest production, but required 12 to 19 days to reach it. At 1 rpm, usual yields of 2.3-2.5 g/kg were obtained in 8-9 days, while only 0.1-0.2 g/kg were produced in static conditions. Hesseltine [26] summarizes the advantages of agitation on mycotoxin production as: a) effective distribution of spore inoculum; b) maintenance of homogeneity and prevention of pellet formation; c) improves aeration; and d) facilitates heat transfer. Two stages are distinguished on research on mycotoxin production by SSF. The first one was developed by Hesseltine’s group from the late 60s through to the mid-70s. They studied production of several mycotoxins using different grains and forages like alfalfa. This method became classic and the main process variables controlled were: 1. Selection of the grains (substrate), and recommended whole or dehulled rice, barley, wheat, corn and soybean. 2. Pretreatment, which includes soak and cooking. Also, light abrasion of the kernel surface (pearl) for barley and wheat and fractionation of the grains of corn and soybean into five or six pieces. 3. Particle size, kept within a limited range to prevent grain agglomeration. 4. Moisture content is important and should be kept low to prevent contamination. 5. Grains should occupy a relatively small volume of the vessel. A second phase was developed during the second half of the 70s, with the work of Demain and co-workers in Boston, USA. This group studied the production of other mycotoxins in the same SSF system [68]. In general terms it can be said that lower yields were obtained and that it became clear that agitation did not favor the synthesis of some toxins [69,70].

Secondary metabolite production in SSF ’

Although the works referred to in the preceding section suggested that high concentrations of secondary metabolites could be produced in solid cultures, there were no further attempts to produce useful secondary metabolites by SSF (with the exception of pigments), until the end of the 80s. In 1987 Kumar and Lonsane [7] reported the production of gibberellic acid by SSF on wheat bran in flasks. A year later our group published the first paper on penicillin production by SSF on inert support, using Raimbault-type column fermenters with forced aeration [4]. This marked the beginning of a new phase of research on secondary metabolites production by SSF. After this several authors have studied production of antibiotics

and other useful secondary metabolites by different SSF systems (Table 2). These works have constituted a second stage of study and interest on the subject, that is characterized by the diversity of secondary metabolites produced and by the use of nontraditional and novel SSF systems. Also, because some of these studies not only optimize the most usual process variables, but perform basic and deeper studies. The analysis of this literature is beginning to form a view of the nature and particularities of idiophase in solid medium, which is described in the next sections.

The SSF system As was previously mentioned, traditional-type SSFs are performed on grains. Our work on secondary metabolites production in solid culture started using SSF on cassava flour (constituted mainly by starch), and studied the synthesis of aflatoxins and gibberellic acid. In that period it was observed that when the carbon source constitutes part of the structure, the physical characteristics of the solid medium Table 2. Recent applications of SSF for the production of antibiotics and other secondary metabolites.

SSF system

Microorganism

Product

Conc.

(Pdd

Time (days)

Ref.

Wheat bran

Gibberellic acid

1217

7

[7,711

Gibberella fujikuroi

Wheat bran

Gibberellic acid

6800

8

[721

Penicillium chrysogenum

Impregnated support Penicillin (sugar cane bagasse)

5-6

[5]

Streptomyces viridifaciens

Sweet potato residues Tetracycline

4720

5

[731

Streptomyces clavuligerus

Barley

Cefamicine

300

10

[741

Achremonium chrysogenum

Barley

Cephalosporine

950

10

[741

Aspergillus parasiticus

Cassava flour

Aflatoxins

Bacillus subtilis

Wheat bran

Iturine

Claviceps purpurea C . fusiformis

Rye support support Rye

Ergot alcaloids

Monascus kaoling

Mantou meal

Pigments

Streptomyces cinnamonensis

Barley/oats

Monensine

Aspergillus oryzae

Wheat and soybean

Pyrazines

Streptomyces aureofaciens

Wheat bran

Aureomycin

Gibberellla fujikuroi, Fusarium moniliformis c

“Production in units of absorbency per ml at 500 nm.

10500

1.2

[66]

3660

2

1751

690 960 2080 0

11

[761

60

8.5

5430 ODU“

[61

250

[771 Rolz et al. [47] 1781

4800

3

[791

99 deteriorate progressively during the culture, reducing mass and energy transfer. Although metabolite synthesis was very fast, cultures could not proceed for long periods (Tomasini, Fajardo, Barrios-Gonzdez, unpublished results). It was thought that the use of an inert support with impregnated liquid medium would solve the problem, since the physical structure would be more or less constant throughout the culture. In practice this SSF system presented additional advantages for basic studies since the same media used in SmF can be used in SSF, the effect of the presence of different compounds can be determined, and the liquid-fermented broth can be extracted (by pressing) and analyzed at any time. Several supports were tested and sugarcane bagasse (pith) stood out, since good growth and production could be obtained, not only for secondary metabolites but also for different enzymes [4,27,80]. Studies on secondary metabolism in SSF were also impulsed by the use of penicillin as a convenient model secondary metabolite. As can be observed in Table 2, studies on secondary products formation, published during the last 8 years, have used different SSF systems; from the adaptation of the koji system used by Hesseltine and co-workers to the inert support with absorbed liquid medium system. Production levels suggest that lower titres are obtained in kojitype SSF systems, and in longer periods. It is possible that extraction processes are more complicated in those systems. High production is observed in impregnated support and in wheat bran systems. However, it is interesting tonote the high titres obtained in sweet potato residues. The culture period reported (5 days) seems very long for a one-support-substrate phase system. It is also convenient to keep in mind the high yields of mycotoxins obtained by the Peoria (Hesseltine) group using agitated grains SSFs (Fig. 4). The microorganisms and the products SSF is defined as a general method for the production not only of mycotoxins, but of antibiotics and other useful secondary metabolites, like plant growth stimulants, alkaloids and pigments. Panorama is also optimistic due to the range of microorganisms that can be used successfully for the production of these compounds. Besides different fungal species, processes using actinomycetes and at least one using a nonfilamentous bacteria (Bacillus) have been reported.

'

Control of secondary metabolism in SSF: environmental factors A careful analysis of the literature mentioned above shows evidence that basic principles, on the factors that govern secondary metabolite production in SSF, are beginning to emerge. Moisture content and water activity Like in the early works on mycotoxins, the new processes confirm the importance of

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Fig. 4. Electron microphotograph showing the adherence of yeast cells to the sugarcane bagasse during a solid culture (Saucedo-Castafiedaet al., Depto. de Biotecnologia, Universidad Aut6noma Metropolitana, MBxico).

moisture content as a fundamental parameter. There is not a study in which this factor has not been optimized, and its importance becomes evident when production changes, under different initial moisture values, are observed. This is an expected fact since water has an important role in enzymes, nutrients and products diffusion through the solid matrix. However, the importance of this variable appears to be more critical for secondary metabolites formation, probably since initial conditions must generate an adequate environment for production, after the rapid growth phase. Water activity (Aw) is related to the concentration of solutes in a liquid medium and represents the unbounded, and therefore available, water. In SSF determination of available water during the culture is an important problem. Moisture content of a solid culture, at any time, is constituted by: 1) available water, 2) bound water, and 3) mycelial constitutive water. As the culture develops this latter fraction becomes greater, while available water decreases. However, available water is an unknown quantity, since there is no quantifying method. Through indirect calculations (estimations and balances) it has been proposed that, in SSFs of one substrate-support phase, growth stops because of exhaustion of the available water, i.e., Aw = 0.85. It is interesting to note that real Aw determination was much higher since it represents the global Aw, including mycelium [63].

101 Initial moisture in different SSF systems Optimal initial moisture contents for growth, depend on the water-holding capacity of the solid medium, and are lowest in grain SSF systems, with values around 35%. In starchy flour systems this parameter is near 50% and in wheat bran between 50 and 60%. The impregnated support system allows the usage of the highest initial moisture contents (up to 78%), with optimum values around 70%. Lotong and Suwanarit [81] have produced red pigments in plastic bags containing rice grains. They observed that pigmentation occurred only at relatively low initial moisture contents ( 2 6 3 2 % ) . It is possible that at these moisture levels nutrients transport or supply during idiophase is slower, giving rise to a slower and more convenient growth rate. In fact the authors report that at higher initial moisture levels higher activities of glucoamylase were obtained. Glucose was rapidly liberated in concentrations of 120 g/l, presumably inhibiting pigmentation. This information suggests that moisture level could be used to control growth rate (or catabolites concentration) in these culture systems. Johns and Stuart [82] confirmed solid culture was superior to liquid fermentation for red pigment production by Monascus purpureus. This result has been attributed to the derepression of pigment synthesis in SSF, due to the diffusion of intracellular pigments into the surrounding solid matrix. In submerged culture, the pigments normally remain in the mycelium due to their low solubility in the usually acidic medium. However, a word of caution is necessary for pigments production after the work of Blanc et al. [83]. The authors demonstrated that, besides the pigments, strains of M. purpureus and M. ruber produce the mycotoxin citrinin in liquid and solid cultures. Moisture content for different microbial groups Different SSF systems have distinct water retention capacity, therefore showing diverse optimal values of initial moisture content (IMC). However, as can be observed in Table 2, in the same SSF system, bacteria (actinomycetes and Bacillus) have a higher requirement for water than fungi. In the wheat bran SSF system, optimal IMC for gibberellic acid production by G. fujikuroi was 60%, while the optimal value for iturine production by Bacillus was 68%. In the case of cephalosporine production optimal IMC in a barley SSF system was 39.5% for Streptomyces clavuligerus, while for the fungus Achremoium chrysogenum was 33%. With tetracyclines the high moisture content that the material (sweet potato residue) can hold is surprising: 68% with an Aw of 0.995. Although there are no reports of an antibiotic of an actinomycete produced in SSF on support, the abovementioned Aw value can be compared with the penicillin production of a solid medium with an Aw of 0.967. Moisture content and water activity: experiments in SSF on support An interesting insight into the nature of secondary metabolism in solid culture is arising from the research performed in SSF on support. Up to what point these

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conclusions can be extrapolated to other SSF systems remains to be studied (Fig. 5). Sato et al. [84]developed a mathematical model of microbial growth in SSF using wood pulp with impregnated liquid medium. Results with Cundidu lipoliticu showed that an increase in specific growth rate (p) is directly related to an increase in moisture of the solid medium, as well as with the relative humidity of the air flow. Later, On01 et al. [63]carried out studies with A. niger in SSF using sugarcane bagasse with absorbed liquid medium. The authors found that, when initial moisture is varied between 40 and 75%, but keeping medium concentration constant (Aw of 0.97),growth rate was not modified (p = 0.4/h).Nevertheless, when Aw (and initial moisture) was modified, between 0.9 and 0.986,with different glucose concentrations, p varied between 0.19 and 0.54/h.The total amount of biomass generated was greater in the more concentrated media, although the yield Yx/s did not vary greatly. The most important impact was on germination time, which varied from 5 to 20 h. It can be said thus, that Aw controls growth in SSF (at least in SSF on support), having a direct effect on growth rate and an inverse effect on germination. Also, that initial moisture content does not have any effect on the growth phase. Although IMC did not have an important effect on trophophase, experiments performed in our laboratory showed that this parameter has an important impact in idiophase. Barrios-Gonzillez et al. [4]showed that IMC has an important influence on the penicillin production level in SSF. The authors modified initial moisture (between 60 and 78%) keeping Aw constant, in a similar way as done by Oriol et al. [63].In cultures with IMCs of 70 and 73%, high production rates were observed during the last part of the fermentations, which allowed the cultures to reach penicillin titres of

Fig. 5. Automatic on-line measuring system of oxygen uptake and carbon dioxide production.This system was designed in Depto. de Biotecnologia, Universidad Aut6noma Metropolitana, MBxico (BaniosGonzilez and Mejia).

103 1,120 pg/g of dry matter, while cultures with lower or higher IMCs only reached 280 pg/g. In the same work, experiments were performed in which nutrients concentration was increased (decreasing Aw), keeping IMC constant in 70%. It was found that unlike trophophase, the use of very concentrated media favors the antibiotic production in SSF. This was an important effect since production increased 5-fold in 2x medium (twice the concentration recommended for SmF). Conversely, the use of concentrated media negatively affected production in SmF. This represents a marked difference between the effect of moisture content (and Aw) in trophophase and idiophase in SSF. The need of concentrated media in SSF for high secondary metabolite production also represents an outstanding difference between physiology in solid and liquid mediums. Very recent studies performed in our laboratory (M. Dominguez, A. Mejia, J. Barrios-Gonzlilez, 1995;unpublished results) widen the view in this field, by demonstrating that IMC and medium concentrations do not control production levels directly. At this point it is important to remember that the SSF on support system is constituted (initially) by three main components: support, water and nutrients. Support and nutrients represent the solids, so to increase the nutrients concentration of the solid medium, keeping a constant moisture value, the support content has to be decreased. The experiments on moisture content performed by Oriol and co-workers were made keeping medium concentration constant (Aw = 0.977) by decreasing bagasse content of the solid medium. To increase the concentration of the liquid medium, the authors decreased support and water content. In this work, it was found that a particular value of IMC (or nutrient content) can be adjusted by varying the other two components (nutrients and support content) in different ways, which resulted in very different penicillin yields. In other words, the same initial moisture (or concentration) value can create different conditions for idiophase. In fact, the different ways in which these two parameters can be adjusted represent different nutrients/bagasse/water combinations. It was observed that when nutrients or moisture content are increased, production can increase or decrease depending on how this variation is compensated with the other two components. However, when bagasse content was decreased, penicillin yields always increased. It was established then that penicillin synthesis in this system is controlled by the proportion of support and the other two components. At this point it is important to note that not all medium components are completely soluble. Solid media with high nutrients concentration and low bagasse content has a very different appearance (dense, humid, soggy) to a solid medium with higher support content (fluffy, dry, low density). Combinations used in these experiments were plotted in a triangle of combinations of nutrient support and water. Conditions of high production were localized in a narrow fringe of low support content (l(t12.5%). In this fringe, maximal titres were detected in a zone low moisture (62%) and high nutrients content (25.5%), as well as in a zone moisture (73-75%) and a medium level of nutrients concentration

104 (12.3-16.25%), which are equivalent to 1.5 and twice the recommended concentration for SmF. Although these initial conditions have low bagasse content in common, it is not clear why such different combinations can create a favorable environment (maybe similar) for penicillin production during idiophase. However, respiration measurements have been discovered to be a very sensitive method for detecting subtle changes in metabolism, and are beginning to throw some light on this and other basic aspects of secondary metabolism in SSF. Respiration studies of the cultures with diflerent components combinations Respiration studies were performed on these fermentations (J. Barrios-Gonzilez, M. Dominguez, A. Mejia, 1995;unpublished results) by means of an automated respirometer [85]. Two kinds of curves were obtained: the derivative (ml CO,/g/min vs. time) and the integrated or accumulative form (ml total COJg) which was calculated from the latter and resembles the growth curve. The accumulative respiration curve (Fig. 6) can be divided into a sharp peak that corresponds to trophophase. When this peak falls, penicillin synthesis starts in all cases. After this, a second peak is formed, which is lower and much wider, presenting a negative slope that falls steadily until, near the end, slopes down until it almost reaches zero at the end of the culture. Penicillin production continues during the second peak. In a typical integrated curve (Fig. 7), the culture starts with a short period of high slope (QC0,-t), followed by a long phase with a lower slope (QC0,-i), which is stable for a period and then decreases steadily. This stage correlates with penicillin production so is identified as idiophase (Fig. 8). The first conclusion drawn from the respiratory experiments is that, like in SmF, production starts when growth (respiratory activity) is limited. This observation agrees with the growth kinetic patterns observed by Kumar and Lonsane [71] on gibberellic acid production on wheat bran SSF. As previously mentioned, penicillin production increases when support content of the solid medium decreases. When respiratory rates (QCO, in ml/min*gdry medium)

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Time (hl Fig. 6 . Respiration kinetics (derivative form) of penicillin SSF. Comparison of high (solid line) and low production conditions (thin line).

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Fig. 7. Time course of C 0 2 production (accumulative form) in SSF by P. chrysogenum with different support (pith bagasse) content: 23,9%(thin line) and 10.3 (solid line).

for tropho- and idiophase were calculated, it became evident that decreasing bagasse content caused a decrease in respiratory rate. This suggests that in solid culture, like in SmF, optimum penicillin production (highest specific production rate) is obtained when the culture grows within a range of low specific growth rates (not lower than O.O15/h) [14]; and that low support content is the way to achieve this in SSF. Conditions of highest penicillin production presented certain common respiratory patterns: 1. The slope of phase 2 (derivative curve) was lower, i.e., tended to form a leveled plateau. 2. CO, evolution rates during idiophase (QC0,i) were always lower than the QC0,i under conditions of lower production. Although numeric values varied among the high production cultures, these QC0,i were always smaller (between 15 and 88% lower) than the ones determined in low production conditions. These results indicate that high production conditions generate metabolic stability during idiophase, with slower respiration rates (and hence growth rates). It appears thus that, under these conditions, nutrients are less exposed and mass transfer

0

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Fig. 8. Time course of penicillin production by P. chrysogenum in SSF with different support (pith bagasse) content: 23.9% (thin line) and 10.3% (solid line).

declines, causing slower nutrient uptake. These initial combinations probably permit an adequate and constant nutrient supply during idiophase, supporting slower but constant growth rates that are more adequate for product biosynthesis. Particle size and packing density Oriol et al. [55] studied growth kinetics of A. niger in SSF on impregnated bagasse. It was found that increases in support particle size caused decreases in growth rate at the end of this growth phase and suggested that this depressive effect could be caused by limitations caused by the intraparticular nutrients diffusion. This effect could favor secondary metabolism since nutrients limitation triggers and maintains idiophase. Barrios-GonzBlez et al. [86] determined the effect of this parameter on penicillin production in a similar SSF system. Although highest titres were reached using the biggest particle size (14 x 1.7 mm; retained in 10 mesh), experiments using washed bagasse showed that the increase in production was due to higher sugar content in that fraction. However, the use of coarse wheat bran (particle size of 0.3-0.4 cm) resulted in an increase of 2.5 times in the yield of gibberellic acid, in relation with smaller particles [7]. The authors indicate that the increase could have been due to the availability of larger void fraction, noncompactness of the sterilized medium, better oxygen transfer and efficient removal of CO, and other volatile products. Packing density of the solid medium is a parameter that is seldom studied in SSF. However, this parameter will be increased in most static reactors that can be used for production scale-up. Barrios-GonzBlez et al. [83] reported a moderate increase in penicillin production (1,25W 1,680 pg/g) by P . chrysogenum Wisconsin 54- 1255 in a densely packed solid-state cultures (0.35 g/ml), using the impregnated bagasse system. The reason for this increment is not clear, but it should be related to the reduction of the interparticular space. Under these conditions, an inhibition of conidiation was also observed. Since sporulation occurs during secondary metabolites formation, it is possible that those two effects could be related. Inhibition of morphogenesis implicates that more intermediates and substrate might be available for secondary metabolites biosynthesis. This hypothesis has also been used to explain yield improvements by agitation in the early mycotoxin work [26]. Another possibility is that contact between mycelia inhibits growth but stimulates secondary metabolism. Aeration and agitation The gas environment may significantly affect the relative levels of biomass and enzyme production [87]. Han and Mudgettt [88] found that levels of 0, and CO, in the gas environment influence pigment production significantly and growth to a lesser extent in SSF. Maximum pigment yields, with Monascus purpurea on rice, were observed at 0.5 atm. of 0, partial pressure in closed pressure vessels. However, high CO, partial pressure progressively inhibited pigment production, with complete

107 inhibition at 1.0 atm. In a closed aeration system with a packed-bed fermenter, oxygen partial pressures ranging from 0.05 to 0.5 atm., at constant CO, partial pressures of 0.02 atm., gave high pigment yields with a maximum at 0.5 atm. of O,, whereas lower CO, partial pressures at constant 0, partial pressure of 0.21 atm. gave higher yields. Experiments performed in our laboratory also indicate that growth is less sensitive to high CO, partial pressures, than penicillin production. In fact these conditions seemed to stimulate germination (unpublished results). Earlier work in our laboratory studied the effect of different air flow rates on aflatoxin production by A. parasiticus in SSF on cassava flour [66]. A moderate positive effect was observed when increasing aeration rates between 0 and 0.3 l/h*g of moistened medium, while no effect was noted on growth of final pH. It was also found that high aflatoxin concentrations were still reached at low aeration rates. The form of the curve was similar to the one obtained by Silman et al. [89], studying aflatoxin production in corn (grains). An important difference is that these authors studied a very different aeration range: 0-0.0024 1h.g of humid corn. It is not clear if different ranges were studied in these two works or if the SSF systems show different aeration needs. In contrast to these findings, ochratoxin A yields in a rotating drum bioreactor were reduced by aeration [67]. It is considered that agitation prevents heterogeneity of the solid medium composition and in mycelial age. Mixing breaks long mycelial nets, generating shorter mycelia in similar physiological stages (this could be useful for secondary metabolite production). Agitation can be used to improve gas exchange and to remove heat from the solid medium. This operation is also a prerequisite for mixing water or other type of additions. As previously mentioned, this can be an essential part of a cooling strategy. Water additions can also be a key operation in prolonging production phase whether the limiting factor is available water or a nutrient depletion. However, it has been reported that some fungi do not tolerate agitation in SSF [12]. Barrios-Gonzilez et al. [86] determined the effect of agitation on penicillin production on impregnated bagasse. The authors did not find a negative effect when P. chrysogenum Wisconsin 54-1255 was used. Similar experiments with the higher producer P. chrysogenum P-2 showed a slight positive effect on product formation (4,000 vs. 5,700 pgJg dry medium) and an important increase in metablic activity: 189 vs. 261 ml CO,/g dry medium, at 96 h. Image analysis indicated that the microbial population was formed by shorter mycelia (approximately 200 vs. 500 p), presenting a higher branching frequency (branching every 67 f 25 vs. 246 f 70 p) [go]. In other words, a higher number of growing points, giving rise to a higher metabolic activity (and probably to a greater secretion surface), as well as greater physiological homogeneity of the culture (Fig. 9). Kumar and Lonsane studied, in a different way, the effect of the degree of aeration on gibberellic acid production in wheat bran SSF. The authors varied the ratio of the volume of the solid medium to the total capacity of the flask. As the ratio increased from 0.024 to 0.1 the yields of gibberellic acid decreased from 1,116 to 916 m a g of dry medium. The 0.024 ratio corresponds to a 1.0 cm depth of the moist medium.

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Fig. 9. View of a pilot reactor (1.6 m3) for SSF; with screws and carriage. This reactor has a maximum working capacity of 1 ton (200 kg dry matter) and can be scaled up to production level (Durand and Cherau, INRA, Dijon, France).

Fig. 10. Pilot fermenter for solid-state cultures. This reactor has been used for the production of penicillin by P. chrysogenum; and for protein enrichment of cassava flour by A. niger (GutiCrrez-Rojas et al. Depto. de Biotecnologia, Universidad Authoma Metropolitana).

109 An important physical factor is pretreatment of the substrate or support. Autoclaving the moist solid medium can cause changes in the substrate and make it more amenable to degradation during fermentation. In the case of inert supports, pretreatment can bring out its full water-holding capacity. It has been reported that gibberellic acid production increased slightly (from 1,023 to 1,032 m a g ) with increased autoclaving time up to 45 min at 121°C. The yield was lower (910 mgkg) with 15 min autoclaving time, indicating insufficient modification of the solid substrate (Fig. 10). Inoculum size

In SSF inoculum must be distributed homogeneously and must be high enough to assure predominance of the strain. In recent work on secondary metabolites production two tendencies can be noted: some inoculate with spores and some inoculate with mycelium from a liquid seed culture. Among the studies in which a spore inoculum was used, the work on alkaloid production on rye grains is interesting in this regard since the effect of inoculum size was determined [76]. Inoculum size varied between 2 x lo6 and 2 x 10' spores/g of dry medium. Results showed that by increasing inoculum size, lag phase (germination) was slightly reduced, while growth rate was increased. However, the highest alkaloid production was obtained with 2 x lo7 spores/g. In the work of tetracycline production with Streptomyces vridifQciens, inoculum size was also optimized, varying this parameter between lo7 and lo9 spores/g of dry medium. In this case too, the highest titres were obtained using lo8 spores/g [73]. It seems that with an actinomycete, with less capacity to colonize the solid medium than fungi, a greater inoculum size is required. The effect of the inoculum type and size on gibberellic acid production on wheat bran was studied by Kumar and Lonsane [46]. Results showed that gibberellic acid production was more or less the same when an inoculum, grown for 7 days in liquid or solid media, was used. However, the yields of this metabolite were reduced by about 33% when inoculum grown in liquid medium was used for 20 h. The maximum production was obtained when 15 or 20% (w/v) liquid inoculum was used, although lower inoculum sizes caused a reduction in yields. The effect of inoculating with different mycelium concentrations (from 0.8 to 2.5 mg of dry biomass/ml) was also tested in cefamycine and cephalosporine production on barley SSF. It was found that antibiotic production increased proportionally to the inoculum size, so 2.5 mg/ml were used throughout the work [74]. In the case of iturine, SSF on wheat bran was inoculated with 3 ml of liquid culture for 15 g of moistened medium, which appears to be a similar proportion to the one used for gibberellic acid. Very recent results in our laboratory indicate that penicillin SSF can also be inoculated with mycelium from liquid culture. Preliminary results indicate that this can even have a positive effect on production and shorten production time.

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Control of secondary metabolism in SSF: nutritional factors (and pathway regulation) In traditional SSFs, modification of the type and concentration of nutrients is done indirectly by changing the grain used. In early work on mycotoxin production on grains, fermentations were not supplemented with additional carbon source. It is possible that a convenient balance of nutrients was obtained by an adequate selection of the grain.

Carbon source In recent research on secondary metabolites production in solid culture, an additional carbon source was often used. The impact of this procedure on production indicates that this is a key parameter. Production of gibberellic acid on wheat bran increased 3.5 times when the solid medium was supplemented with 20% of soluble starch, although higher concentrations had a deleterious effect [71]. The use of a fed-batch SSF with intermittent feeding of soluble starch powder had a moderate positive effect of 18% on production [7]. A subsequent report [91] described a 47% increase in product yield over batch culture using feeds of corn starch. The reason for this increase was that production continued for another day. This is a very important result since high productivity process requires high production rates for as long as possible. Tetracycline production on SSF with sweet potato residues showed a 270% increase when the medium was enriched with 10% soluble starch [73]. It is important to note that the addition of 10% glucose caused a 60% decrease in the antibiotic titres. Supplementation with 5% sucrose had a positive effect on alkaloid production in SSF on rye grains. To increase alkaloid production on SSF on support (sugarcane bagasse) a careful optimization of the medium was done by statistical response surface methods. The results of the analysis identified sucrose (250 g/l) as one of the key factors, together with triptophan (precursor) and phosphate (limiting nutrient; phosphate regulation) [76]. Conversely, supplementation with glucose, KH,PO, and MgSO, did not affect iturin production on wheat bran SSF with a strain of Bacillus subtilis [75].

Pathway regulation Studies in liquid medium have shown that carbon source can also have negative effects on the synthesis of secondary metabolites. In the case of penicillin, glucose depresses the synthesis of the antibiotic, repressing transcription of enzymes of the pathway (ACV synthetase and isopenicillin N synthetase) [92]. The use of SSF on support has permitted the initiation of studies on how regulatory mechanisms operate in solid medium. This information is not only important from the basic point of view, it could find applications in the design of production processes as well as in the

111

development of overproducing strains. Initially it seemed likely that secondary metabolite synthesis in solid medium could be subregulated due to limitations in mass transfer in this system. However, our studies on aflatoxin production in cassava SSF showed that when the concentration of phosphate and ammonium in the solid medium were reduced, aflatoxin production increased proportionally [66]. Since the synthesis of these metabolites is regulated by phosphate and ammonium, these results insinuated that the biosynthesis of aflatoxins was regulated, in solid medium, in a similar way as in liquid medium. On the other hand, Ramesh and Lonsane [93] reported what they called the ability of SSF (wheat bran) to minimize catabolic repression on a-amylase of Bacillus lichenformis. The authors found that enzyme production in SmF decreased drastically when soluble starch concentration of the medium was increased from 0.2 to 1%. In comparison, enzyme production in SSF increased 29-fold when starch concentration was increased in wheat bran SSF. Later, the authors [94] observed that the addition of 1% glucose to a liquid culture completely suppressed production, while in SSF 15% glucose slightly stimulated production. Similar results were obtained by Solis et al. [28] on pectinases production by A. niger using SSF on impregnated bagasse. In SmF an initial glucose concentration of 3% caused a strong decrease of enzymatic activity, while in SSF, concentrations up to 10% had a stimulatory effect on production. Although these results give the impression of a much greater regulatory threshold in solid culture, sugar consumption kinetics suggest that these differences are more related to the capacity to consume sugars in solid culture (very rapid uptake rate), and not to differences in regulatory thresholds. In SSF 90% of the sugar was consumed in 24 h, while in SmF this proportion of the sugars was consumed in 96 h. Taking advantage of the facility of SSF on support to use exactly the same medium composition as in liquid culture, and to analyze the fermentation broth, our group has compared the effect of glucose on penicillin biosynthesis in SSF and in liquid culture. Regulatory thresholds were estimated by carefully correlating glucose uptake kinetics with the initiation of penicillin biosynthesis. Results (SmF: 28-20 gll; SSF 3G14 g/l) indicated that penicillin synthesis is also regulated by carbon catabolite regulation in SSF and at similar thresholds as the ones observed in SmF [951. On the other hand, the high concentrations of penicillin that can be obtained in solid medium (see section on: The strain and the production level) ih this culture system must represent a problem to the fungus. The relatively low diffusion rates in the solid medium can cause an accumulation gradient of antibiotics near the mycelium. Feedback regulation of penicillin G biosynthesis (by penicillin V) in SSF was recently studied in our laboratory. Results indicated that this mechanism is also active in SSF [96], and suggested that, as was found in liquid medium [23], the regulatory threshold of a strain is directly related to its productivity. However, the thresholds estimated in SSF appear to be higher than in liquid medium (Fig. 11). These results strongly suggest that mechanisms which regulate the biosynthetic pathway of secondary metabolites in SSF also play a role in solid culture.

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Fig. 11. Laboratory scale rotatory reactor for SSF, designed and built in Depto. de Biotecnologia, Universidad Autonoma Metropolitana, MCxico. This fermenter is used to study the effect of mixing and water and/or nutrients additions on penicillin fermentation (Barrios-Gonzdez, Mejia and Miranda).

Fig. 12. General view of a pilot scale reactor (50 I total volume) for asceptic SSF. This reactor has been used for gibberellic acid production by fed-batch.

113 Nitrogen source Concentration and type of nitrogen source is important in growth and production media in SmF. Although during the early work on mycotoxin production no nitrogen source was added to the medium, recent work on secondary metabolites production in different SSF systems has shown the importance of doing so. Yang and Ling [73] optimized tetracycline production in SSF on sweet potato residues. One of the parameters that contributed most to increased production was nitrogen source. The authors started by decreasing C/N ratio from 65 to 20 with (NH,),SO,, increasing production from 48 to 322 pdg. They fixed the concentration of this compound in 0.5% (1% had a negative effect) and combined it with 20% of an organic nitrogen source (peanut flour or wheat bran). This had a synergic effect since tetracycline production increased to 2,000 pdg. It is also interesting to note that tetracyclines spectrum varied when the nitrogen source was changed. When NH,NO, was used, tetracycline and chlortetracycline were produced in a 59.4 and 28.1% proportion. When the combined nitrogen source was used these percentages changed to 18.3-38.6%. For alkaloid production in SSF on rye, a mixture of urea and ammonium sulfate was used to control pB during the culture. The studies on SSF on inert support found ammonium sulfate as the best nitrogen source, and was an important factor for production increase (2,500 pdml) [76]. Limiting nutrient In liquid medium secondary metabolism starts when growth is limited by the exhaustion of a key nutrient. The studies on respiration kinetics and penicillin regulation described above, together with the observations made in the works of aflatoxins (phopsphate and/or ammonium concentration caused a decrease in yields); and alkaloids (low phosphate, a key factor) formation, indicate that secondary metabolism is triggered, in SSF, by the same stimuli as in liquid medium. Precursor effect In liquid culture, the addition of a precursor (biosynthetic pathway intermediate) that is rate limiting, during antibiotic and other secondary metabolites formation, brings about an increase in production and/or directs the synthesis preferentially towards one of the metabolites of the family. No specific studies on this subject have been performed in solid culture, however, the effect of triptophane on alkaloid synthesis mentioned before, as well as the increase in tetracycline production by the addition of methionine indicates a precursor effect. In our research on penicillin production, phenylacetic acid has been used as a precursor for penicillin G. The HPLC analysis have not shown evidence that related penicillins are being synthesized. All this indirect evidence suggests that precursor effect is also manifesting in solid

114 culture. This can have practical importance in a commercial process since the formation of other penicillins, or related compounds in the case of other secondary metabolite production, is negative for its economy.

The strain and production level The development of highly productive microbial strains is a prerequisite for efficient biotechnological processes. Up to the present time, strain improvement was mainly based on induced mutagenesis. Mutation and selection has been responsible for the impressive increases in penicillin titres (several 100-fold), while only during the last few years has genetic recombination based on parasexual crosses and protoplast fusion became an additional practicable strategy [97]. Although recombinant DNA techniques (genetic engineering) provide an enormous potential for strain improvement in industrial microorganisms, its impact is just beginning to be felt. Basic cloning systems have now been developed for various microorganisms used in industrial processes [98]. Special strain for SSF

One of the major positive aspects of SSF is that metabolites are, in many cases, produced at much higher yields than by liquid fermentation (SmF) [4,5,25,99]. However, there are only a few reports on comparative studies, and these works have been carried out with low-yielding strains, which are closer to the wild-types than to the hyperproducing mutants used in modem SmF industry. In fact there is a lack of information regarding what kind of strains are needed for SSF processes. Overproducing strains, developed for liquid medium, might not have the expected performance in a SSF process. This can cause not only an underestimation of the potential of SSF for certain applications, but could be a serious handicap for this culture method, since most or all of the overproducing strains have been developed for SmF. Shankaranand et al. [lo01 observed discrepancies in the levels of a-amylase produced by strains of Bacillus in SSF in relation with SmF. The authors concluded that cultures that are good producers in SmF cannot be relied upon to perform well in SSF. A recent study of our group [5] showed that, as in SmF, the strain is a fundamental element in the production level that can be obtained in SSF. It was found that higher yielding strains, developed for SmF, also tend to be the highest producers in SSF. This agrees with our results on regulation of penicillin biosynthesis in SSF, since it implicates that mutations that allow high production in liquid medium are also useful to overproduce in solid medium. Strains efSiciency in SSF

In the same work [5] parameters were defined which allow quantitative evaluation of

115 the efficiency of different strains to produce in SSF. Relative production was defined as the ratio = peak production in solid divided by peak production in liquid. This parameter can be roughly interpreted as the number of times production in solid is greater that production in liquid culture (if >l). Relative productivity was defined in a similar way. With this tool it was possible to determine that higher yielding strains (for SmF) tend to be less efficient in SSF, that is these strains show relative productions of 0.1-4, while lower yielding strains can show relative productions of 17. This implicates that there is one or several characteristics, probably not related to the biosynthetic pathway, that allow high production in SSF (besides the ones needed to produce in liquid medium), which are more frequently found in low producing strains. These characteristic(s) are unknown but are expressed as high relative production. Since this value was higher in lower yielding strains, it is possible that these characteristic(s) have been lost during the genetic improvement programs.

Strain improvement for SSF Although higher yielding strains (for SmF) tend to be less efficient in solid culture, some mutants derived from these strains displayed very good performance in solid medium (high relative production). This is the basis of the method described to select high producing mutants particularly suited for SSF. In this work mutant strains were generated that produced up to 10,500 pg of penicillin per g of dry medium, representing production increases, in relation to parental strains, of between 500 and 640% [ 5 ] . This means that isolating mutants from (SmF) high-yielding strains, and selecting as performed in this work, is an efficient and rapid way of generating penicillin (and probably other secondary metabolites) overproducers particularly suited for SSF. The characteristics that allow a high relative production do not seem to be related with antibiotic biosynthetic pathway since are only expressed in SSF. Neither do these characteristics seem to be related to fast and abundant growth in solid medium since P. chrysogenum P2-4 (the highest producing mutant obtained) reached peak production in 130 h, while lower producing mutants like ASW8-20 (8,750 pg/g) in 85 h and Wisconsin 54-1255-6 (4,530 pg/g) in only 60 h, with much greater apparent growth. This can also be noted in the study on alkaloid production by SSF. Six strains of Cluviceps were evaluate in SmF and production of the two best was optimized in SSF on impregnated support. Alkaloid production of C. fusiformis (ATCC 26019) was high in both culture systems (solid and liquid), even though growth of this strain in SSF was very poor (4.7 g of dry biomassfl vs. 17.5 g/l) as compared with the other strain (C. purpurea 1029~).It is noteworthy that mentioning that C. fusiformis practically did not grow on rye grains and displayed the slowest apical growth rate in petri dish [76]. On the other hand it is surprising that Ohno et al. [75] produced 3,660 pg of ituridg in 2 days, in a wheat bran SSF system, using a wild strain of B. subtilis

116 isolated from compost. More surprising is the comparison of that titre with production reached in SmF: 150 pdml in 5 days (even though a different medium was used since this SSF system is not suitable for comparative experiments). From these figures a relative production of 24.4 can be calculated. This results support our earlier conclusion: that strains closer to the wild-type tend to show a higher relative production. The characteristics that allow good performance in SSF are not known, but this information would provide a deep understanding of microbial adaptation to solid medium. From an applied point of view, this knowledge would be very helpful to design more direct rational selection methods as well as genetic engineering strategies for genetic improvement of these strains. In studies on pectinases production by SSF on coffee pulp, Antier an co-workers [loll found that the use of low water activity as a selective factor, for mutants resistant to 2-deoxiglucose (2DG), seems to select a special kind of mutants named dgrAw96. These are pectinase hyperproducing mutants for SSF (228 U/g dry pulp). Mutant strains that overproduced pectinases in SmF were also obtained using a similar selective medium (pectin + 2DG) but with high Aw (0.999). The fact that the hyperproducing mutants for SSF show a rapid and vigorous growth in SSF attracts our attention. As mentioned before, in secondary metabolism this characteristic, although desirable, is not usually associated to high production. At the present time it is not clear if this difference is due to the fact that it is a catabolic enzyme or because the SSF system used is a one phase support-substrate. Since the support is also the substrate, during the course of the fermentation Aw decreases sharply, since available water is decreasing while sugars are constantly dissolving in it. In the system of support with absorbed medium, available water is probably increasing while sugars and other nutrients concentration decreases. In this way the characteristics needed in a strain could be different for different SSF systems. It can be speculated that in SSF the microorganism senses the solid environment and reacts producing special enzymes that are more efficient in a medium with restricted diffusion. It is possible that membrane structure varies also to adapt to a medium with an air-water-support interface, with different conditions for nutrient uptake and product secretion. Constant protection against osmotic pressure and ionic toxicity might be another particular feature of SSF. All this suggests the existence of some regulatory mechanisms of solid medium adaptive genes (Fig. 12).

Concluding remarks SSF could become an alternative production method for antibiotics, growth promoters and other secondary metabolites. It is in the production of such high value products that advances in SSF are most likely to be made. Only from the economic returns from such products will incentives and resources be available to carry out research and development to solve problems associated with SSF (Mitchell and Lonsane, 1992).

117

However, without a deeper knowledge of the nature of secondary metabolism in solid medium it will not be possible to obtain the systems full production potential. Moreover, there will be little foundation from which process engineers can design and build large-scale systems. Research performed over the last 8 years indicates that different kind of secondary metabolites can be produced by SSF: antibiotics, phytohormones, food grade pigments, alkaloids, etc., using fungi, actinomycetes and Bacillus. Very high concentrations have been obtained in SSF on inert support and on wheat bran, although other agricultural products or byproducts can also be used. Moisture content of the solid medium stands out as a key factor in secondary metabolites production. Experiments in SSF on support indicate that control of idiophase can be achieved by manipulating the support content of the solid medium. This operation appears to control metabolic activity (growth rate) during production phase. Hopefully, findings in this system will eventually be applied in other SSF systems. Basic studies on nutritional factors have shown that physiology in SSF has several similarities with the known behavior in liquid medium. Hence, similar strategies must be adapted to avoid regulation by carbon, nitrogen or phosphate. High production in SSF also requires supplementation with an adequate carbon source as well as with inducers and precursors. It has been found that special strains are needed for SSF processes, but that these strains can be obtained by modifying the overproducing mutants used in SmF. Moreover, the characterization of these strains could provide valuable information about the particularities of the microbial adaptation to a solid environment. Further research is needed to asses ways in which the productivity of SSF may be increased. Areas deserving attention include: the use of fed-batch or continuous plugflow modes of operation. Analysis of performance of SSF bioreactors to identify criteria for successful scale-up and to permit effective process control. The latter is particularly important given the lengthy nature of these cultures (Johns, 1992).

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