MAIN CHARACTERISTICS AND APPLICATIONS OF SOLID SUBSTRATE FERMENTATION

N. Pérez-Guerra, A. Torrado-Agrasar, C. López-Macias and L. Pastrana*

Department of Biochemistry, Genetics and Immunology, University of Vigo, Spain. E-mail: pastrana@uvigo.es

KEYWORDS

Solid substrate fermentation; solid matrix; fungi; bioreactor design.

ABSTRACT

Solid substrate fermentation (SSF) has been usually exploited for the production of value-added products (antibiotics, alkaloids, plant growth factors, etc), biofuel, enzymes, organic acids, aroma compounds and also for bioremediation of harzardous compounds, biological detoxification of agro-industrial residues, nutritional enrichment, biopulping, biopharmaceutical products, etc. This technology has gained renewed attention from industry because it has become a more attractive alternative to liquid fermentation for many productions. Thus, SSF was found to produce a more stable product, with less energy requirements, in smaller fermenters and smaller volumes of polluting effluents. This paper aims to present an overview of the main characteristics and applications of SSF, as well as their advantages and disadvantages compared to submerged liquid fermentation (SLF). The effect of the main variables in SSF processes and important aspects related to design of SSF bioreactors are also discussed.

Solid state fermentation

Definition

Solid-state fermentation (SSF) processes can be defined as “the growth of microorganisms (mainly fungi) on moist solid materials in the absence of free-flowing water” [1,2]. These processes have been used for the production of food, animal feed, and both pharmaceutical and agricultural products.

Substrates that have been traditionally fermented by solid-state include a variety of agricultural products such as rice, wheat, millet barley, grains, beans, corn and soybeans. However, non-traditional substrates which may also be of interest in industrial process development include an abundant supply of agricultural, forest and food-processing wastes (such as wheat bran and soy grits (flakes remaining after extraction of oil)).

1.        Comparative studies between submerged liquid fermentation (SLF) and SSF claim higher yields and other advantages for products made by SSF [1, 3-7]:

2.        Similar or higher yields than those obtained in the corresponding submerged cultures.

3.        The low availability of water reduces the possibilities of contamination by bacteria and yeast. This allows working in aseptic conditions in some cases.

4.        Similar environment conditions to those of the natural habitats for fungi, which constitute the main group of microorganisms used in SSF.

5.        Higher levels of aeration, especially adequate in those processes demanding an intensive oxidative metabolism.

6.        The inoculation with spores (in those processes that involve fungi) facilitates its uniform dispersion through the medium.

7.        Culture media are often quite simple. The substrate usually provides all the nutrients necessary for growth.

8.        Simple design reactors, with few spatial requirements can be used due to the concentrated nature of the substrates.

9.        Low energetic requirements (in some cases autoclaving or vapour treatment, mechanical agitation and aeration are not necessary).

10.     Small volumes of polluting effluents. Fewer requirements of dissolvents are necessary for product extraction due to their high concentration.

11.     The low moisture availability may favour the production of specific compounds that may not be produced or may be poorly produced in SLF.

12.     In some cases, the products obtained have slightly different properties (e.g. more thermotolerance) when produced in SSF in comparison to SLF.

13.     Due to the concentrated nature of the substrate, smaller reactors in SSF with respect to SLF can be used to hold the same amounts of substrate.

In the same way, SSF has some disadvantages when compared with the submerged-liquid cultures [1, 3-5, 7]:

1.        Only microorganisms that can grow at low moisture levels can be used.

2.        Usually the substrates require pre-treatment (size reduction by grinding, rasping or chopping, homogenisation, physical, chemical or enzymatic hydrolysis, cooking or vapour treatment).

3.        Biomass determination is very difficult.

4.        The solid nature of the substrate causes problems in the monitoring of the process parameters (pH, moisture content, and substrate, oxygen and biomass concentration).

5.        Agitation may be very difficulty. For this reason static conditions are preferred.

6.        Frequent need of high inoculum volumes.

7.        Many important basic scientific and engineering aspects are yet poor characterised. Information about the design and operation of reactors on a large scale is scarce.

8.        Possibility of contamination by undesirable fungi.

9.        The removal of metabolic heat generated during growth may be very difficult.

10.     Extracts containing products obtained by leaching of fermented solids are often viscous of nature.

11.     Mass transfer limited to diffusion.

12.     In some SSF, aeration can be difficult due to the high solids concentration.

13.     Spores have longer lag times due to the need for germination.

14.     Cultivation times are longer than in SLF.

Selection of the microorganisms

The ability of the microorganisms for growing on a solid substrate is a function of their requirements of water activity, their capacity of adherence and penetration into the substrate and their ability to assimilate mixtures of different polysaccharides due to the nature, often complex, of the substrates used.

The filamentous fungi are the best-adapted microorganisms for SSF owing to their physiological, enzymological and biochemical properties. The hyphal mode of fungal growth gives the filamentous fungi the power to penetrate into the solid substrates. This also gives them a major advantage over unicellular microorganisms for the colonisation of the substrate and the utilisation of the available nutrients. In addition, their ability to grow at low water activity (a_w) and high osmotic pressure conditions (high nutrient concentration) makes fungi efficient and competitive in natural microflora for bioconversion of solid substrates [8]. From a practical point of view, the vegetative growth is preferred over the sporulation.

However, bacteria and yeasts have also been used in traditional cultivation in SSF processes [9]. Bacteria have been used for enzymes production, composting, ensiling and some food processes (e.g. sausages, Japanese natto, fermented soybean paste, Chinese vinegar) [8, 10, 11]. Yeasts have been mainly used for ethanol production and protein enrichment of agricultural residues [12-17].

Support and nutrient source

In SSF, two types of process can be distinguished depending on the nature of the solid phase. In the first and the most used, the solid serves both as a support and a nutrient source. These substrates are heterogeneous water insoluble materials from agriculture or by-products from food industry, which have an amylaceous or ligno-cellulosic nature (grains and grain by-products, cassava, potato, beans and sugar beet pulp) [18]. For this last characteristic, these should be pre-treated to convert the raw substrate into a suitable form to increase its utilisation by the microorganism. This includes [19]:

·         size reduction by sifting, grinding, rasping or chopping.

·         damage to outer substrate layers by grinding, pearling or cracking.

·         chemical or enzymatic hydrolysis of polymers.

·         supplementation with nutrients (phosphorus, nitrogen, salts) and setting the pH and moisture content, using a mineral solution.

·         cooking or vapour treatment for macromolecular structure pre-degradation and elimination of major contaminants.

In the second, an inert support (sugar cane bagasse, hemp, inert fibres, resins, polyurethane foam and vermiculite) is impregnated with a liquid medium, which contains all the nutrients (sugars, lipids, organic acids, etc). This strategy is less used, but it reports some advantages. The use of a defined liquid medium and an inert support with a homogenous physical structure improves controlling and monitoring the process and the reproducibility of fermentations. In any case, the use of inert supports presents economical disadvantages [20].

In both cases, the success of the process is directly related to the physical characteristics of the support, which favour both gases and nutrients diffusion and the anchorage of the microorganisms [8]. From a practical point of view, the following physical characteristics of the solid matrix must be taken into account because of their influence on the development of SSF [19]: particle size and shape, porosity and consistency of the material.

ENVIRONMENTAL FACTORS AFFECTING MICROBIAL GROWTH AND PRODUCT SYNTHESIS IN SSF

Environmental factors such as water activity, moisture content, temperature, pH, oxygen levels and concentrations of nutrients and products significantly affect microbial growth and product formation.

Water activity and moisture content of the substrate

The role of the water content of the substrate has been widely described and reviewed by different authors [21-26, 1].

Moisture content is a critical factor on SSF processes because this variable has influence on growth and biosynthesis and secretion of different metabolites [27-28]. A lower moisture content causes reduction in solubility of nutrients of the substrate, low degree of swelling and high water tension. On the other hand, higher moisture levels can cause a reduction in enzyme yield due to steric hindrance of the growth of the producer strain by reduction in porosity (interparticle spaces) of the solid matrix, thus interfering oxygen transfer [29].

As the optimal value of moisture content depends on both the microorganism and the solid matrix used, for economical production, the microorganism should be grown in optimal moisture levels either for maximising the growth or metabolite production (enzymes, organic acids, etc) depending on the application.

Generally, the water content of the substrate oscillates between 30 and 75%. Lower values can induce the sporulation of the microorganism, whereas higher levels can reduce the porosity of the system, which can produce oxygen transfer limitation, and increase the risk of bacterial contamination. During fermentation, the water level of the substrate can change due to evaporation and microbial activity. In general, the result of all these processes is the loss of humidity, being necessary to add water using humidificators or applying a water saturated air flow.

The water requirements of microorganism must be better defined in terms of water activity (a_w) rather than water content of the solid substrate [3]. Water activity is defined as the relationship between the vapour pressure of water in a system and the vapour pressure of the pure water. From a microbiological point of view a_w indicates the available or accessible water for the growth of the microorganism. The water activity affects the biomass development, metabolic reactions, and the mass transfer processes [21-22].

Although water activity is a function of the concentration of the solutes, in those systems in which solutions are adsorbed in a matrix, the values of a_w also depend on the physical structure and the chemistry nature of the matrix. The adequate value of a_w depends on both the product and the requirements of the microorganism [22]. Generally, a_w for metabolite production is higher than for growth.

Mass transfer processes: aeration and nutrients diffusion

In SSF, the mass transfer processes related to gases and nutrients diffusion are strongly influenced by the physical structure of the matrix and by the liquid phase of the system [30, 21, 3]. Raghavarao et al. [30] described two kinds of phenomena of mass transfer; one at micro-scale and other at macro-scale outside the cells. The first deals with the mass transfer into and out of the microorganism cells. The second includes more factors: the bulk air flow into and out of the bioreactor, natural convection, diffusion and conduction trough the substrate, the materials of the bioreactor, the shear damage of the microorganism and the integrity of the substrate particles.

1.        Gases diffusion: The aeration has essentially two functions: (1) oxygen supply for aerobic metabolism and (2) removal of CO₂, heat, water vapour and volatile components produced during the metabolism [21]. The exchange of O₂ and CO₂ between the solid and the gas phase takes place at both inter-particular and intra-particular level. This depends on those factors that increase the contact surface between the phases [1]: void fraction of the matrix, pore and diameter size of the particle, degree of mixture and depth of the matrix, additional aeration generated by forced step of sterile air and agitation and moisture level of the substrate. In general, the gases diffusion increases with the pore size and decreases with the reduction of the diameter due to substrate packaging [31].

2.        Nutrients diffusion: It occurs at an intraparticular level and includes both the diffusion of nutrients toward the cells and the hydrolysis of solid substrates by the microbial enzymes [31]. This last point is an important aspect in SSF because the most part of the substrate is water insoluble [30].

In substrates with a small pore size, the resistance to the intraparticular mass transference increases with the diameter of the particle and the degradation of the substrate occurs mainly at the outer surface.

Nutrient diffusion processes are especially important in bacterial and yeast SSF. They are not so critical for fungi cultures because the mycelium can better penetrate the solid matrix.

Temperature

The increase in temperature in SSF is a consequence of the metabolic activity when the heat removal is not enough. This affects directly spores germination, growth and product formation. The temperature level reached is a function of the type of microorganism, the porosity, the particle diameter and the depth of the substrate [3,21,30].

Heat transfer in SSF is very low because of the limited heat transfer capacity of the solid substrates used. The overall heat transfer depends on the rates of the intra- and inter-particle heat transfer and the rate at which heat is transferred from the particle surface to the gas phase [3].

Control of temperature is more difficult in SSF than in submerged fermentation. Thus, the control methods used in SLF are not suitable for SSF. In an industrial context, monitoring and controlling this variable is critical for scaling up [22]. Conventionally aeration is the main method used [3,30] to control the temperature of the substrate. Because high aeration rates can reduce the water activity of the substrate by evaporation, water saturated air is usually used [30]. The agitation of the fermentation mass can also help to control the temperature.

pH

The measurement and control of this variable in SSF is very difficult. Nevertheless, the substrates employed in SSF usually has buffering effect due to their complex chemical composition. In these cases, the control of pH is not necessary. When this variable must be controlled, buffering solutions are added as liquid phase, but this strategy can be inadequate when the process is scaled-up. Another possibility to control the evolution of the pH consists on adding a mixture of sources of nitrogen with opposite influence on the evolution of the pH in such a way than ones counteract the effect of the others. In this sense, ammonium salts have been used in SSF in combination with urea or nitrate salts due to the respective effects of acidification and alkalization of the former and the latter [3,32].

Applications of SSF

Socio-economic applications of SSF offer the potential of significantly raising living standards with only a low technology input requirement [8]. Several authors have reviewed the different applications of solid-state fermentation [3,33]. SSF is briefly associated with the production of traditional fermented foods such as “koji”, Indonesian “tempeh” or Indian “ragi”. SSF has also been used for the production of high added value compounds (such as enzymes, organic acids, biopesticides, biofuel and flavours). In the last years, new applications of SSF in the environmental control have been developed including bioremediation and biodegradation of hazardous compounds and the detoxification of agro-industrial residues. Table 1 shows some examples of SSF processes in economical sectors of agro-industry, environmental control and fermentation industry [3,33].

Some engineering considerations of solid-state fermentations

Classification and design of SSF bioreactors

Many bioreactors have been traditionally used in SSF processes. These can be mainly classified as: packed-beds, rotating drums, gas–solid fluidized beds, stirred aerated beds, rocking drums and tray bioreactors. From a practical point of view, SSF processes could be operated in batch, fed-batch or continuous modes, although batch processes are the most common [30].

Papers deal with bioreactor design for SSF are scarce. However, due to some problems associated with solid beds like poor mixing, heat transfer, characteristics and material handling, SSF bioreactor systems are yet to reach a high degree of development. In this way, some aspects must be taking into account for the design of a solid-state bioreactor system [30,80]:

The wide variety of solid substrates employed in SSF, which have important differences such as composition, size, mechanical resistance, porosity and water holding capacity.

The SSF bioreactors must be constructed with a strong material, which must be anticorrosive and non-toxic to the process organism. It should also have a low cost.

The entry of contaminants into the process as well as the uncontrolled release of the process organism into the environment must be avoided by using filters on outlet air stream and by a careful design of seals and filtration of the inlet air stream.

Other important aspect to be considered during the construction of a bioreactor is the effective regulation of aeration, mixing and heat removal. This could avoid the problems related to an ineffective heat removal, evaporative loss of water from the substrate bed and thermal gradients, which affect the yield and quality of the desired product. In the same way, the control of operational parameters (temperature, water activity and oxygen concentration) and the maintenance of uniformity within the substrate bed could be as effective as possible.

A bioreactor system could be designed to facilitate the substrate preparation, its sterilization and of the biomass after product recovery, inoculation, loading and unloading of the bioreactor as also product recovery. In this sense, a novel and efficient design of integrated solid matrix bioreactor called the PLAFRACTOR^TM has been recently reported. This bioreactor is a computer-controlled compact device wherein all the operations described above could be possible [81].

The effect of the shear forces generated by mixing of both the substrate and the microorganism should be also considered. In this way, it has observed that these forces can damage the penetrative hyphae and affect the gel substrate [30,82]. In this sense, other modern SSF bioreactor is being manufactured and marketed by M/s Fujiwara, Japan. This reactor consists of a rotating bed in the form of a bucket with provision of mixing of the substrate by ribbon-shaped baffles. In this equipment, operations like substrate sterilization and inoculation are automated. Slow rotation of the helical screws ensures minimal damage to growing hyphae and also provides an effective mixing of the substrate bed. This coupled with a slow rotation of the basket allows the elimination of temperature gradients within the substrate bed. The forced aeration of humid air from the bottom allows the alleviation of oxygen gradients without lowering the moisture content of the bed [83].

The shrinking of the substrate bed pulling it away from the bioreactor walls is a problem that can occur in the case of microbially degradable substrates [84]. As cause of this, the air can flux through the gaps between the bed and walls. This undesirable channelling of the air could be avoided by using a non-degradable matrix by the microorganism [85].

CONCLUDING REMARKS

In general, SSF is a well-adapted and cheaper process than SLF for the production of a wide spectra of bioproducts (animal feed, enzymes, organic acids, biopulp, aroma compounds, antibiotics, compost, biopesticide, biofertilizer, etc). It has been reported that in many bioproductions, the amounts of products obtained by solid state fermentation are many-fold higher than those obtained in submerged cultivations. In addition, the products obtained have slightly different properties (e.g. more thermotolerance) when produced in SSF and SLF. Therefore, if SSF variables are well controlled and the purity of the product is defined, this technology may be a more competitive process than is commonly thought.

There are, however, some important problems associated to solid state fermentation: designs for upscaling, and control of operations (mainly heat transfer and cooling) and fermentation variables (mainly pH and temperature). In addition, the diffusion of products through the solid media lead to both extraction processes (in which concentrated impurities could also be obtained) and purification steps. This contributes to an increase in recovery costs.

In this respect, some advances have been made in the development of adequate and efficient devices for heat and mass transfer control, mainly in scale-up reactors. In the same way, interesting approaches have been used by some researchers to characterise oxygen transfer, to measure some variables of the process (temperature, moisture content, pH, etc) and to model microbial growth.

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