Aerobic microbial transformation of solid materials or "Solid Substrate Fermentation" (SSF) can be defined in terms of a solid porous matrix which can absorb water with a relatively high water activity. The solid/gas interface should be a good habitat for the fast development of specific cultures of moulds, yeasts or bacteria, either by isolated or mixtures of species. The mechanical properties of the solid matrix should stand compression or gentle stirring as required for a given fermentation process. This requires small granular or fibrous particles, which do not tend to break or stick to each other. The solid matrix should not be contaminated by inhibitors of microbial activities and should be able to absorb or contain available microbial foodstuffs such as carbohydrates (cellulose, starch, sugars) nitrogen sources (ammonia, urea, peptides) and mineral salts.

Traditional fermentations are typical examples of SSF:

- Japanese koji, which uses steamed rice as solid substrate inoculated with solid strains of the mould Aspergillus oryzae.

- Indonesian tempeh or Indian ragi which use steamed and cracked legume seeds as solid substrate and a variety of non toxic moulds as microbial seed.

- French "blue cheese" which uses perforated fresh cheese as substrate and selected moulds, such as Penicillium roquefortii as inoculum.

- In addition to traditional fermentations new versions of SSF have been invented. For example, it is estimated that nearly a third of industrial enzyme production in Japan which is made by SSF process and koji fermentation has been modernised for large scale production of citric and itaconic acids.

- Composting which was produced for small-scale production of mushrooms has been modernised and scaled up in Europe and United States. Also, various firms in Europe and USA produce mushroom spawn by cultivating aseptically Agaricus, Pleurotus or Shii-Take on sterile grains in static conditions.

Generally, most of the recent research activity on SSF is being done in developing nations as a possible alternative for conventional submerged cultures which are the main process for pharmaceutical and food industries in industrialised nations.

SSF seems to have theoretical advantages over LSF. Nevertheless, SSF has several important limitations. Most of the processes are commercialised in Southeast Asian, African, and Latin American countries. Nevertheless, a resurgence of interest has occurred in Western and European countries over last 10 years.

- Potentially many high value products as enzymes, metabolites, antibiotics, could be produced in SSF. But improvements in engineering and socio-economic aspects are required because processes must use cheap substrate locally available, low technology applicable in rural region, and processes must be simplified.

- The greatest socio-economical potential of SSF is the raising of living standards through the production of protein rich foods for human consumption. Protein deficiency is a major cause of malnutrition and the problem will become worse with further increases in the world population. Two ways can be explored for that:

- Production of protein-enriched fermented foods for direct human consumption. This alternative involves starchy substrates for its initial nutritional calorific value. Successful production of such food will require demonstration of economical feasibility, safety, significant nutritional improvement, and cultural acceptability.

- The second alternative consists to produce fermented products for animal feeding. Starchy fermented substrates with protein enrichment could be fed to monogastric animals or poultry. Fermented lignocellulosic substrates by increasing in the fibre digestibility could be fed to ruminants. In this case, the economical feasibility should be decisive in comparison to the common model using protein of soybean cake, a by-product of soybean oil.

Since 15 years, the Orstom group investigated on solid fermentation process for improving protein content of cassava and other tropical starchy substrates using fungi (especially from Aspergillus group) in order to transform starch and mineral salts into fungal proteins (Raimbault, 1981).

- Protein enrichment of Cassava and starchy substrates

- Production of organic acids or ethanol by SSF from starchy substrate and Cassava

- Digestibility of fibres and lignocellulosic materials for animal feeding

- Degradation of caffeine in coffee pulp and ensiling for conservation and detoxification

- Enzymes and fungal metabolites production by SSF using sugarcane bagasse

Bacteria, yeasts and fungi can grow on solid substrates, and find application in SSF processes. Filamentous fungi are the best adapted for SSF and dominate in research works.

Bacteria are mainly involved in composting, ensiling and some food processes (Doelle et al., 1992). Yeasts can be used for ethanol and food or feed production (Saucedo et al., 1992a, 1992b). But filamentous fungi are the most important group of microorganisms used in SSF process owing to their physiological, enzymological and biochemical properties. The hyphal mode of fungal growth and their good tolerance for low Aw and high osmotic pressure conditions make fungi efficient and competitive in natural microflora for bioconversion of solid substrates.

Koji and Tempeh are the two most important applications of SSF with filamentous fungi. Aspergillus oryzae is grown on wheat bran and soybean for Koji production, which is the first step of soy sauce or citric acid fermentation. Koji is a concentrated hydrolytic enzymes required in further steps of the fermentation process. Tempeh is an Indonesian fermented food produced by the growth of Rhizopus oligosporus on soybeans. The fermented product is consumed by people after cooking or toasting. The fungal fermentation allows better nutritive quality and degrades some antinutritional compounds contained in the crude soybean.

The hyphal mode of growth gives also the filamentous fungi the power to enter into the solid substrates. The cell wall structure attached to the tip and the branching of the mycelium ensure firm and solid structure. The hydrolytic enzymes are excreted at the hyphal tip, without large dilution like in the case of LSF, that makes very efficient the action of hydrolytic enzymes and allows penetration into most solid substrates. Penetration increases the accessibility of all available nutrients within particles.

In general, substrates for SSF are composite and heterogeneous products from agriculture or by-products of agro-industry. This basic macromolecular structure (e.g. cellulose, starch, pectin, lignocellulose, fibres etc.) confers the properties of a solid to the substrate. The structural macromolecule may simply provide an inert matrix within which the carbon and energy source (sugars, lipids, organic acids) are adsorbed (sugarcane bagasse, inert fibres, resins). But generally the macromolecular matrix represents the substrate and provides also the carbon and energy source.

The most significant problem of SSF is the high heterogeneity, which makes difficult to focus one category of hydrolytic processes, and leads to poor trials of modelling.

Lignocellulose occurs within plant cell walls, which consists of cellulose microfibrils embedded in lignin, hemicellulose and pectin. Each category of plant material contains variable proportion of each chemical compound.

Pectins are polymers of galacturonic acid with different ratio of methylation and branching. Exo-and endo pectinases and demethylases hydrolyse pectin in galacturonic acid and methanol. Hemicellulases are divided in major three groups: xylans, mannans and galactans. Most of hemicellulases are heteropolymers containing two to four different types of sugar residue.

Lignin represents between 26 to 29% of lignocellulose, and is strongly bounded to cellulose and hemicellulose, hiding them and protecting them from the hydrolase attack. So the lignocellulose hydrolysis is a very complex process. Effective cellulose hydrolysis requires the synergetic action of several cellulases, hemicellulases and lignin peroxydases. But lignocellulose is a very abundant and cheap, natural, renewable material, so a lot of works were dedicated to microorganisms breakdown, especially fungal species.

Starch is another very important and abundant natural solid substrate. Many microorganisms are capable to hydrolyse starch, but generally the efficient hydrolysis requires previous gelatinisation. Some recent works concern the raw (crude or native) starch like it occurs naturally.

Within the plant, cell starch is stored in the form of granules. During the process of gelatinisation, starch granules swell when heated in the presence of water, which involves the breaking of hydrogen bonds, especially in the crystalline regions. Many microorganisms can hydrolyse starch, especially fungi, which are suitable for SSF application involving starchy substrates. Glucoamylase, a-amylase, b-amylase, pullulanase and isoamylase are involved in the processes of starch degradation. Mainly a-amylase and glucoamylase are of importance for SSF.

Microorganisms generally prefer gelatinised starch. But large quantity of energy is required for gelatinisation, and it would be attractive to use organisms growing well on raw (ungelatinised) starch. Different works are dedicate to isolate fungi producing enzymes able to degrade raw starch, as has been done by Soccol et al (1991), Bergmann et al. (1988) and Abe et al. (1988).

In our lab we developed many studies concerning SSF of cassava, a very common tropical starchy crop, in the view of upgrading protein content, both for animal feeding using Aspergillus sp. Initial protein content (1-4 %) could be increased until 18-20 % Dry Matter basis.

Recently Soccol using selected strains of Rhizopus biotransformed cassava in starchy fermented flours containing 10-12% of good protein, comparable to cereals. Such biotransformed Cassava flour can be used as cereal substitute for breadmaking until 20% without sensible change for the consumer.

Biomass is a fundamental parameter in the characterisation of microbial growth. Its measurement is essential for kinetic studies on SSF. Direct determination of biomass in SSF is very difficult due to problems of separation of the microbial biomass from the substrate. This is especially true for SSF processes involving fungi, because the fungal hyphae penetrate into and bind the mycelium tightly to the substrate. On the other hand, for the calculation of growth rates and yields it is the absolute amount of biomass, which is important. Methods that have been used for biomass estimation in SSF belong to one of the following categories.

Direct measurement of exact biomass in SSF is a very difficult question. For that we can consider the global stoechiometric equation of the microbial growth:

- Respiratory metabolism

Oxygen consumption and carbon dioxide release result from the respiration, the metabolic process by which aerobic microorganisms derive most of their energy for growth. As carbon compounds within the substrate are metabolised, they are converted into biomass and carbon dioxide. Production of carbon dioxide causes the weight of fermenting substrate to decrease during growth, and the amount of weight lost can be correlated to the amount of growth that has occurred.

The measurement of either carbon dioxide evolution or oxygen consumption is most powerful when coupled with the use of a correlation model. If both the monitoring and computational equipment is available then these correlation models provide a powerful means of biomass estimation since continuous on-line measurements can be made. Other advantages of monitoring effluent gas concentrations with paramagnetic and infrared analysers include the ability to monitor the respiratory quotient to ensure optimal substrate oxidation, the ability to incorporate automated feedback control over the aeration rate, and the non-destructive nature of the measurement procedure.

- Production of extracelluIar enzymes or primary metabolites

Associated extracellular enzymes are another metabolic activities, which may be produced. It is observed frequently a good correlation between mycelial growth and organic acid production, which can be measured by the pH measurement or a posteriori correlated by HPLC analysis on extracts. In the case of Rhizopus, Soccol (1992) demonstrated a close correlation between fungal protein (Biomass) and organic acids (citric, fumaric, lactic or acetic).

Protein content: The most readily measured biomass component is protein. The Folin method is more sensitive and allowed a greater dilution of the sample which avoided interference from the starch in the substrate.

Glucosamine: A useful method for the estimation of fungal biomass in SSF is the glucosamine method. This method takes advantage of the presence of chitin in the cell walls of many fungi. Chitin is a poly-Nacetylglucosamine. Interference with this method may occur with growth on complex agricultural substrates containing glucosamine in glucoproteins (Aidoo et al, 1981).

Ergosterol: Ergosterol is the predominant sterol in fungi. Glucosamine estimation was therefore compared with the estimation of ergosterol for determination of the growth of Agaricus bisporus (Matcham et al, 1985).

Physical measurement of biomass: Peñaloza (1991) used another physical parameter to evaluate mycelial growth, based on the difference in the electric conductivity between biomass versus the substrate. Good correlation with biomass was obtained and a model was proposed. Recently Auria et al. (1990) monitored the pressure drop in a packed bed during SSF of Aspergillus niger on a model solid substrate consisting of ion exchange resin beads. Pressure drop was closely correlated with protein production. Pressure drop is a parameter, which is simple to measure and can be measured on-line. Further studies are required to determine whether the use of pressure drop in monitoring growth in forcefully aerated SSF bioreactors is generally applicable. An interesting point of this physical technique resides in the fact that it is sensible to the conidiation: early conidiophore stage makes the pressure drop drastically and a breaking point can be easily observed.

On the other hand, in the production of protein enriched feeds, the protein content itself is of greater importance than the actual biomass concentration, and the variation in biomass protein content during growth becomes less relevant.

Overall, oxygen uptake and carbon dioxide evolution methods are probably the most promising techniques for biomass estimation in aerobic SSF as they provide on-line information. The monitoring and computing equipment is relatively expensive and will not be suitable for low technology or rural applications. None method is ideally suited to all situations so the method most appropriate to the particular SSF application must be chosen on the basis of simplicity, cost and accuracy. The best choice could be to cross two or three, or more, techniques for measurement of various parameters, and the total balance could be highly correlated to the actual biomass.

Environmental Factors

Environmental factors such as temperature, pH, water activity, oxygen levels and concentrations of nutrients and products significantly affect microbial growth and product formation. ln submerged stirred cultures environmental control is relatively simple because of the homogeneity of the suspension of microbial cells and of the solution of nutrients and products in the liquid phase.

SSF process can be defined as microbial growth on solid particles without presence of free water. The water present in SSF systems exists in a complexed form within the solid matrix or as a thin layer either absorbed to the surface of the particles or less tightly bound within the capillary regions of the solid. The optimum Aw for growth of a limited number of fungi used in SSF processes was at least 0.96 whereas the minimum growth Aw was generally greater than 0.9. This suggests that fungi used in SSF processes are not especially xerophilic. The optimum Aw values for sporulation by Trichoderma viride and Penicillium roqueforti were lower than those for growth (Gervais et al. 1988). Maintenance of the Aw at the growth optimum would allow fungal biomass to be produced without sporulation.

Stoechiometric global equation of respiration is highly exothermic and heat generation by high levels of fungal activity within the solids lead to thermal gradients because of the limited heat transfer capacity of solid substrates. In aerobic processes, heat generation may be approximated from the rate or CO₂ evolution or O₂ consumption. Each mole of CO₂ produced during the oxidation of carbohydrates released 673 Kcal. That is why it is of high interest to measure CO₂ evolution during a SSF process, because it is directly relied to the risk of elevation of temperature.

Heat removal is probably the most crucial factor in large scale SSF processes , and conventional convection or conductive cooling devices are inadequate for dissipating metabolic heat due to the poor thermal conductivity of most solid substrates and result in non acceptable temperature gradients. Only evaporative cooling devices provide sufficient heat elimination. Although the primary function of aeration during aerobic solid state cultivations was to supply oxygen for cell growth and to flush out the produced carbon dioxide, it also serves a critical function in heat and moisture transfer between the solids and the gas phase. The most efficient processes for temperature control consist in evaporating water, what needs in return to complete the loss to avoid desiccation.

The pH of a culture may change in response to microbial metabolic activities. The most obvious reason is the secretion of organic acids such as citric, acetic or lactic acids, which will cause the pH to decrease, in the same way than ammonium salts consumption. On the other hand, the assimilation of organic acids which may be present in certain media will lead to an increase in pH, and urea hydrolysis result in an alcalinisation.

Aeration fulfils four main functions in solid state processes, namely (i) to maintain aerobic conditions, (ii) for carbon dioxide desorption, (iii) to regulate the substrate temperature and (iv) to regulate the moisture level. Solid state process allows free access of atmospheric oxygen to the substrate; aeration may be easier than in submerged cultivations because of the rapid rate of oxygen diffusion.

Conclusion

SSF is a well-adapted process for cultivation of fungi on natural vegetal materials, which are breakdown by excreted hydrolytic enzymes. In contrast with LSF, in SSF processes, water related to the water activity is a limiting factor, both parameters no involved in LSF where water is in large excess. On the other hand, oxygen is a limiting factor in LSF but not in SSF where aeration is facilitated by the porous and particular structure and high surface contact area which facilitate transfers between gas and liquid phases.

SSF are aerobic processes where respiration is a predominant processes for energy supply to the mycelium; but it can cause severe limitation of the growth when heat transfer is not efficient enough causing rapid elevation of the temperature.

Is the reason why it is so important to study and control respirometry in SSF? We developed a laboratory technique to measure CO₂ and O₂ on line in SSF. A special lecture will be dedicated to the theory, modelling and basic concept of respirometry. Also it will be organise training cessions at the lab, to practice respirometric measurement and kinetics analysis.

(For Literature References, please refer to the original article)