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17.21 Solid state fermentations

In solid state fermentations, microbial growth occurs at the surface of solid substrates and the main difference from submerged liquid fermentation is the quantity of fluid involved. As described above, the biomass grows in suspension in the culture medium in liquid fermentations; whereas a solid state fermentation has a continuous gas phase between the particles or fragments of solid substrate, with a minimum of visible water.

The majority of the water in the solid state system is absorbed within the moist substrate particles and most of the space between particles is filled by the gas phase, although some water droplets may be visible between substrate particles and the particles themselves may be covered in a water film. This type of fermentation is one type of the more general solid substrate fermentation which can vary from processes that involve suspending the solid substrate particles in a continuous liquid phase (rather like food digestion in a stomach or rumen) through to processes (such as trickle filters) in which the liquid phase flows around and through an immobilised substrate.

Solid state fermentations can be divided into two groups according to the physical state of the substrate:

  • low moisture solids fermented with or without agitation, and
  • columns packed with solid substrate which is fermented as liquid is trickled through the column (with or without recirculation).

The substrate usually provides a rich and complex source of nutrients, which may not need to be supplemented. Mixed substrates of this sort are ideal for filamentous fungi able to form mycelia that can grow on and through particulate substrata producing a variety of extracellular enzymes. Traditional substrates for solid state fermentations are various agricultural products like cereal straw and other plant litter, and rice, wheat, maize, and soybean seeds. The majority of solid state fermentations involve filamentous fungi, though yeasts and bacteria can also be cultivated in this way; most are obligate aerobes (though there is no reason why the system should not be operated anaerobically) and the process may feature pure cultures of specific organisms or mixtures of several organisms.

Solid state fermenters are still the method of choice to produce spores of some (most?) fungi for use as biological control agents. It is difficult to produce good fungal spores for biological control purposes in submerged culture (Verticillium lecanii is a notable exception). These features are dealt with in Section 17.19 (CLICK HERE to view now).

A major problem with solid state fermentations is that they are not easy to control and this can make it difficult to meet regulatory requirements. Composting processes can release noxious gases (ammonia and volatile sulphides in particular) and, in addition, aerial spores produced by the organisms involved may be potential health hazards as allergens.

Traditional examples of solid state fermentations are:

  • the formation of compost,
  • mushroom cultivation (and, in a separate process, the production of starter cultures or mushroom spawn),
  • leavening of bread dough, and
  • mould ripening of cheeses, and other food products, such as salami and soy sauce.

What are effectively solid state fermentations also play a role in the production of chocolate and coffee. In these cases the fermentation is responsible for the preparation of the ‘bean’, to separate it from the fruit flesh and mucilage in cocoa; or the cherry and mucilage in coffee and impart flavour.

Cocoa products from the cacao tree, Theobroma cacao, are used in the food, chemical, pharmaceutical and cosmetic industries. The plant originated in the Amazon basin but is now an important cultivated crop worldwide in tropical regions. Cacao fruits are oval pods, 20 to 30 cm long and weighing in the region of 0.5 kg. They grow directly from the trunk and main branches of the tree and are usually harvested by hand. Each pod contains about 40 seeds (the cocoa ‘beans’, though they are not true beans) embedded in a thick pectinaceous pulp. Most of the initial processing is done on the farm. Harvested pods are cut open with a machete and the contents of pulp and cocoa seeds scooped out by hand. The rind is discarded, but pulp and seeds are piled in heaps for several days. During this time, the seeds and pulp undergo what is called ‘sweating’; this is a natural microbial fermentation that liquefies the thick pulp so that it trickles away, leaving the cocoa seeds behind to be collected. Two simultaneous processes occur during their fermentation:

  • microbial activity in the mucilaginous pulp produces alcohols and organic acids as by-products of microbial metabolism, which also liberates heat, raising the temperature to about 50°C;
  • complex biochemical reactions occur within the seed cotyledons due to the diffusion of metabolites from the microorganisms and the rising temperature.

The fermentation features a succession of a range of yeasts, lactic acid bacteria, and acetic acid bacteria. Rapid growth of yeasts occurs in the first 24 hours (including Saccharomyces cerevisiae, Kloeckera apiculata, Candida bombi, C. rugopelliculosa, C. pelliculosa, C. rugosa, Pichia fermentans, Torulospora pretoriensis, Lodderomyces elongiosporus, Kluyveromyces marxianus and K. thermotolerans). The most important roles of the yeasts are:

  • breakdown  of citric acid in the pulp leading to a change in pH from 3.5 to 4.2, which allows growth of bacteria,
  • ethanol production (substrate for acetic acid bacteria),
  • formation of organic acids (oxalic, succinic, malic and acetic),
  • production of some organic volatiles which are probably precursors of chocolate flavour in the cocoa ‘bean’,
  • secretion of pectinases to break down the pulp; pectin is the major plant polysaccharide responsible for the viscosity of the pulp and the yeast pectinases are needed to reduce viscosity of the pulp allowing fluid to drain away to increase aeration (required by acetic acid bacteria).

Unfermented cacao seeds do not produce chocolate flavour (their flavour is similar to that of raw potatoes) and it has not been possible to mimic the production of chocolate flavouring by treating fresh cacao seeds with hot alcohol and organic acids. Flavour development has an absolute requirement for the complex physical and organic biochemistry that occurs during the fermentation. The fermented beans are sun-dried before transfer to processing plants which roast the beans, then separate cocoa butter (used to make chocolate bars) from the solid matter (powdered to make cocoa for beverages and cooking) (Schwan, 1997; Schwan & Wheals, 2004; and see http://www.cocoatree.org/).

Coffee undergoes a similar fermentation, and for similar reasons. Neither coffee beans nor cocoa beans are really beans. Strictly, ‘beans’ are the seeds of plants belonging to the legume family (like faba beans, butter beans, etc.). Cocoa beans are cacao seeds, formed in a seed pod made of several carpels, while coffee beans are seeds formed in a fruit made from a single carpel (called a drupe) which has an outer fleshy part surrounding a central shell (the pit or stone) with one or more seeds inside. Common fruits that are drupes include cherry, apricot, damson, nectarine, peach, plum, mango, olive, date, coconut and coffee. Several species of the bushy tree Coffea are grown for the beans, but Coffea arabica has the best flavour. ‘Robusta’ beans (Coffea canephora var. robusta) are usually grown on land unsuitable for Coffea arabica. The tree produces red or purple fruits, which are cherry-like drupes containing two seeds (the coffee ‘beans’). Harvesting is again done by hand; freshly harvested berries are washed and soaked in water, then most of the berry flesh is removed by a machine that has a roller with a roughened surface that scours away the pulp under a stream of water. In the second stage the coffee ‘beans’ are fermented in large water containers; fermentation dissolves any remaining fruit flesh and also removes a pectinaceous film surrounding the coffee seeds, which is not water soluble. Fermentation takes approximately two days and is important to give the coffee its rich aroma and special flavour. When fermentation is complete, the coffee beans are washed, sun-dried for five or six days and finally this ‘pergamino coffee’ is ready for distribution and/or export (see http://www.hollandbymail.com/coffee/index.html).

According to Wikipedia ‘…After water, tea is the most widely-consumed beverage in the world…’ (http://en.wikipedia.org/wiki/Tea). Tea is made from the processed leaves, leaf buds, and internodes of Camellia sinensis:

  • the most tender leaves and buds are plucked from the bushes by hand,
  • the leaves are then withered to reduce moisture content by up to 70%,
  • limp (withered) leaves are rolled mechanically, which breaks open the leaves and creates the twisted wiry looking tealeaves,
  • when rolling is complete, the leaves are spread on tables to provide good aeration to the released enzymes. The leaves turn progressively from green, through light brown, to a deep brown, and the oxidation takes from between 30 to 180 minutes at about 26°C.

This enzymatic oxidation, which is generally called tea fermentation, is an essential key step in the processing, during which chlorophyll breaks down, tannins are released, and polyphenols in the tea leaf are enzymically oxidised and condensed to form the coloured compounds that contribute to the flavour, colour and strength qualities of the final beverage. The tea industry maintains that the term fermentation is a misnomer, because the enzymes concerned in the oxidation are endogenous to the plant and microbes are not involved. However, we’re not convinced; given the prevalence of endophytic (Section 13.19) and epiphytic (Section 13.20) fungi!

Recently, the solid state approach has been developed for the commercial production of extracellular enzymes and other fungal products, and fungal spores for use as inoculum for biotransformations and, especially, as mycopesticides. The main advantage of this approach for microbial pesticide production is that the process can be done by individual farmers or local communes. Local production avoids some of the major problems associated with large scale commercial production facilities such as poor stability and short shelf-life, and associated storage and long distance shipping problems. The locally-produced mycopesticide can be much cheaper and the formulation can be optimised for local environmental conditions (Capalbo et al., 2001; Pandey, 2003).

The fermenters used for large scale solid state fermentations are called bioreactors. They may be as simple as a plastic bag or an open tray of some sort, including even simple stacks of compost, which may be so large as to require heavy plant for mixing. More ‘engineered’ equipment might consist of stacked arrangements of trays through which temperature and humidity controlled air is circulated, or rotary drum type bioreactors possibly including some additional agitation (Mitchell, Krieger & Berovic, 2006).

A practical generalised approach for production of fungal spores for mycopesticide preparations is a two-stage fermentation procedure in which the fungus is first grown in liquid medium batch culture that is used to seed a solid substrate (usually autoclaved seeds or cereal grains, often rice) for a solid state fermentation that enables conidiation.

At the end of the solid state fermentation the conidiated substrate is air dried before extraction of the conidia using a MycoHarvester, after which the purified spores are dried, suitably formulated into the final product and packaged.

MycoHarvesters are two stage devices consisting of a rotating drum agitator for the overgrown substrate and a spore extractor section comprising four or more stainless steel chambers containing ‘air cyclones’. These cyclones are rapidly rotating air vortices driven by a powerful fan that draws air through the equipment. The agitator releases spores mechanically, and substrate debris and spores are sucked into the cyclones.

The cyclones effectively centrifuge the particles so that air containing the conidia is drawn out of the machine into a collector, while the large substrate particles fall out of the cyclone to the sides and bottom of the unit (see http://www.dropdata.net/mycoharvester/index.htm).

Updated December 17, 2016