1.3.1 Characteristics of Solid-State Fermentations
Solid-state fermentation (SSF) processes are defined as any fermenta- tion process taking place on or within solid substrates or supports in the absence or near absence of free water (Pandey et al. 2000). However, during fermentation of cocoa beans and during wet processing of coffee beans, small quantities of free water are present during fermentation. Thus, these fermentations may be characterized as a semi-solid-state fermentation process (Couto et al. 2001). This kind of fermentation process is very poorly described in the literature. However, it shares more features with SSF than with submerged liquid fermentations, which may be defined as any fermentation process occurring “on” solids dissolved (or submerged) in plenty of free water (Murthy et al. 1993; Ray and Sivakumar 2009). One of the main characteristics is that growth and metabolism of the microorganisms during semi-solid- state fermentations occur on the solid substrate and not in the liquid. Thus, the focus of this chapter will be on the SSF process.
1.3.2 Solid-State Fermented Foods
Some of the first known uses of SSF were, in fact, within the manu- facture of foodstuffs, dating back to 2000 bc (bread making in Egypt) and 3000 bc (soy sauce koji making in China) (Pandey et al. 2008).
1.3.2.1 Determination of Microbial Physiology and Biochemistry in SSF
In SSF involving filamentous fungi, the determination of fermentation kinetics (i.e., biomass production), substrate uptake, and metabolite production is difficult, as cells, substrate, and metabolites are main- tained within the solid matrix. Thus, in these types of SSF, growth kinetic data are generally provided by the use of indirect methods, comprising measurements of cell components, such as DNA, glucos- amine, ergosterol and protein, or measurements of metabolic activity, such as respirometry and microcalorimetry (Bellon-Maurel et al. 2003; Rodriguez-Leon et al. 2008). However, in SSF involving bacteria and yeasts, the growth of cells in single cultures can be quantified by standard microbial isolation and detection methods; that is, sampling, pretreatment, dilution, inoculation on plates, incubation, and plate
counting or by quantitative real-time PCR (qPCR). For mixed cul- ture fermentations, the same approach can be used but aided by spe- cies or group specific isolation media and subsequent identification, or the use of species-specific qPCR (Rantsiou et al. 2008; Falentin et al. 2010). For the estimation of substrate uptake and metabolite produc- tion, not only high pressure liquid chromatography (HPLC) analysis of extracts can be applied (Raimbault 1998), but also new innovative techniques, such as aroma sensing and infrared spectrometry, may be used (Bellon-Maurel et al. 2003). Moreover, as will be described later in this chapter, microbial growth and physiology on solid surfaces may be determined at a single cell level using fluorescence microscopy and image analysis techniques.
1.3.2.2 Control of Growth Conditions in SSF SSF is a complex and
heterogeneous process, due to the fact that cells, nutrients, and prod- ucts occur in three different physicochemical phases; for example, the carbon and energy source is in the solid phase, oxygen is in the gas phase, and microbial cells and their metabolic products are in the liq- uid phase surrounding the solids. In contrast, a submerged fermenta- tion is a more homogeneous process, due to the homogeneity of the suspension of cells, and the solution of nutrients and products, in the liquid phase. Thus in SSF, the fermentation problems may arise due to lack of mixing, resulting in gradients of temperature, oxygen, water, pH, nutrients, and products. These problems render measurement and control of crucial growth conditions, such as temperature, pH, water activity, and oxygen levels, difficult (Raimbault 1998).
1.3.2.3 Cultivation Technique and Bioreactor Design Although it is possi-
ble to operate SSF in fed-batch and continuous modes, batch processes are most commonly used. Mainly two bioreactor types are nowadays available for SSF batch processes; that is, the packed bed (with forced aeration) and the tray (without forced aeration) bioreactor (Mitchell et al. 2000). To the best of our knowledge, the packed-bed bioreactor, as defined above, is not used for industrial food fermentations. As will be seen in later chapters of this book, a tray-like bioreactor, that is, the tray fermentation system (Allison and Rohan 1958; Allison and Kenten 1963), has been developed for controlled fermentation of cocoa in Ghana (Amoa-Awua 2014, Chapter 3 of this book). The tray bioreactor
consists of a chamber; the size of which can vary from as small as an incubator to as large as a room, containing a number of trays. The indi- vidual trays, often of a depth of 10 cm in the case of cocoa fermenta- tion (Allison and Kenten 1963; Nielsen et al. 2007) and the bottoms of which are usually perforated, may be made of plastic, metal, or wood as it is the case with fermentation of cocoa (Allison and Kenten 1963; Nielsen et al. 2007). The substrate bed in a tray may be turned by hand, due to the difficulty in automating the handling of trays, or it may simply be left static. The growth conditions are controlled by regulating the temperature and humidity of the air which is blown through the chamber (Mitchell et al. 2000). Another system, which is widely used for controlled fermentation of cocoa in Brazil, Indonesia, and Malaysia, is the box bioreactor (Wood and Lass 1985). This system may also fall into the category of a tray bioreactor as a less-controlled heap fermen- tation system, but it may be considered to contain only one tray, the bottom of which is not perforated, and with a much larger depth of the substrate bed than the ordinary tray bioreactor, thus rendering control of growth conditions more difficult. Alternatively, it may be regarded as a packed-bed bioreactor without forced aeration.
In small-scale production sites, coffee is fermented in wooden drums or boxes, whereas in large coffee production facilities the wet fermentation takes place in concrete tanks (Wrigley 1988). As for the tanks, the substrate bed may be turned by hand, or it may be left static. The growth conditions during the wet processing of coffee beans may be controlled by regulating the temperature of the water. As men- tioned previously, however, the coffee fermentation process is virtually not described in the scientific literature from a technological point of view, leaving plenty of opportunities for further investigation.
1.4 Methods to Study the Microbial Ecology and Chemistry