Fermentation

General Considerations
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
Micro-Organisms 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.

Substrates

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.