Solid State Fermentation (SSF)

The term solid state fermentation (SSF) is used to refer to culturing microorganisms on moist solid substrates, which can be used as a source of carbon or nitrogen, without a free aqueous phase. These fermentation conditions require less water, thus more closely resemble the natural environment to which the microorganisms are adapted (Pandey et al., 2000). This method is capable of achieving high yields of fermented products (Holker et al., 2004). Due to increase the scale of SSF, several strategies associated with SSF process have been improved, although validation at a large scale is lacking (Mitchell et al., 2000).

Compared to submerged fermentation, SSF has similar yields, however it has the following advantages: (1) no excess water or limited water use, (2) inexpensive sterilization, (3) simple aeration and simple reactor design, (4) reduced operational cost and low energy requirements, (5) inexpensive product recovery and drying, (6) more natural conditions for lignin degrading fungi to grow, (7) lower risk of contamination from bacteria, (8) uniform dispersion of inoculum through the medium, (9) lower levels of pollutants and lower solvent used (Lee, 1997; Toca- Herrera et al., 2007) However, the disadvantages of SSF over submerged fermentation were also reviewed and reported by Toca-Herrera et al. (2007) to be as follows: (1) limited to microorganisms which can grow at low moisture levels, (2) substrate required for pretreatment (chopping, grinding, etc), (3) monitoring

conditions (pH, moisture, oxygen) and biomass yield are difficult, (4) high volume of inoculums is required, (5) risk of contamination by other fungi, (6) lack of aeration and heat transfer, (7) requires a longer period of cultivation.

Figure 17 shows the detail of SSF. Following fungal sporulation fungal hyphae develop into a mycelial mat, while the aerial hypha allow gaseous exchange to occur. The metabolic activities shown mainly occur close to the substrate surface and within the pores and the products, such as enzymes, metabolite and biochemical products are mostly released during fermentation into the solid matrix, therefore these products need to be extracted for further use (Holker and Lenz, 2005).

Figure 17. Micro-scale schematic model of SSF (Holker and Lenz, 2005)

Temperature, pH, oxygen levels, substrate and moisture content are several important aspects that need to be considered in the optimization of SSF (Holker et al.

2004; Lee, 1997). The optimum growth temperature of most basidiomycetes is 28— 30˚C (Jennison et al. 1955), with the maximum temperature for some at 35 to 40˚C (Bis‘ko et al., 1983). Most white rot fungi are mesophiles, with a temperature optimum between 15 and 35˚C. P. chrysosporium, is a moderate thermophile (Lee, 1997), and the specific growth rate of Coriolus hirsutus dramatically increases with increasing temperature up to 30˚C (Emelyanova, 2005) and some thermophilic fungi have optimum growth between 45-50˚C (Wojtczak et al., 1987).

The particle size of SSF media is an important factor, and increasing the solid surface area affects the degree of penetration of the substrate by the fungus. Reducing particle size of aspen wood significantly influenced lignin loss and digestibility rate (Reid, 1985). Kerem et al. (1992) observed that reducing particle size of cotton stalks to less than 2mm was very effective in breakdown by a combination of two mixed cultures of the white rot fungi P. chrysosporium and Pleurotas ostreatus. However, varying particle size in the range of 1 to 8 mm had no effect on wheat straw degradation by P. chrysosporium or Dichomitus squalens (Zadrazil and Brunnert, 1982).

Moisture content is also important for fungal growth. A moisture film sufficient to cover the substrate but which can still allow air to circulate through the substrate is used for solid substrate fermentation (Lee, 1997). The optimum water content to degrade lignocellulose in straw was 3ml water per gram straw (Zadrazil and Brunnert, 1982), while delignification of aspen wood by Phlebia tremellosa was optimal using 2ml water per gram wood (Reid, 1985).

Aeration is also important to biomass production; it has been found in Coriolus hirsutus that the effective oxygen supply was 0.9-1.5g O2 per liter per hour

(Emelyanova, 2005). Increasing O2 pressure beyond 1 atm (100 kPa) did not increase the rate of lignin degradation by P. chrysosporium (Reid and Seifert, 1980), while Phlebia tremellosa degraded lignin in aspen wood at a partial pressure of O2 as low as 7 kPa (Reid, 1985).

Optimum pH for growth varies depending on the microorganism. The optimum pH for basidiomycetes is between 5-6 (Bis‘ko, et al., 1983). Most white rot fungi grow best at a pH of between 4 and 5. The optimum pH for growth also varies with substrate composition (Lee, 1997). For example, the optimum radial growth of Coriolus hirsutus in agar culture was found to be pH 5-6, Emelyanova, 2005). pH also influences enzyme production, particularly cellulase (Pardo and Forchiassin, 1999). The optimum pH for cellulase production is 5.5 for Aspergillus niger, while pH 5.5-6.5 is optimum for β-glucosidase production by Penicillium robrum. Enzyme production is dependent on nutrient availability and increases when there is an addition of a nitrogen source, such as ammonium sulfate or nitrate. Similarly, phosphorus availability affects fungal growth and metabolism (Gark and Neelkantan, 1982). Phenolic compounds and sugar levels also influence enzyme production (Kumar et al., 2008).

The degradation of wheat straw SSF is also influenced by nutrient amendment. Lignin degradation by many white rot fungi is maximal under nitrogen supplementation (Lee, 1997). The addition of urea stimulates lignin degradation of wheat straw by Coprinus sp. (Yadav, 1987) and greater ligninolytic activity is also influenced by the addition of C, particularly glucose, as shown with Trametes versicolor in a straw fermentation system (Zafar et al., 1996). Glucose, ammonium sulphate and molasses were used as nutrient supplements at various stages of T.

versicolor SSF. The effects of supplying nitrogen on the production of lignocellulose degrading enzymes (e.g carboxymethyl cellulase, xylanase, laccase, manganese peroxidase) by white rot basidiomycetes (e.g Pycnoporus coccineus, Cerrena maxima, Funalia trogii, Trametes pubescens, Coriolopsis polyzona, Pleurotus ostreatus) have been examined by Elisashvili et al. (2008). They compared wheat straw with other substrates (e.g tree leaves, apple peels, banana peels). In general, the addition of nitrogen sources such as KNO3, (NH4)2SO4, and NH4NO3 in P. ostreatus wheat straw SSF lowers specific enzyme activities (e.g. xylanase, laccase or manganese peroxidase), however, it has a positive impact on protein production (Elisashvili et al. 2008). In contrast, the degradation of wheat straw lignin by P. chrysosporium is more effective under nitrogen-starved conditions compared to Coriolus versicolor (Zafar et al., 1989 and Fenn and Kirk, 1981). It has been reported that cellulose dropped from 42.3% to 38% after 35 days of incubation. Coriolus versicolor degraded cellulose rapidly within the period of 14 and 21 days before levelling off beyond this period and the lignin content declined from 12.4 to 7.2% after 35 days of incubation (Zafar et al., 1989).