Gallic acid (3,4,5-trihydroxy benzoic acid) is a phenolic compound, mainly used in the pharmaceutical industry for manufacturing trimethoprim, an antibacterial agent (Kar and Banerjee, 2000). Gallic acid is also used in food industries as a substrate to produce a potent antioxidant, propilgallate. Tannase or tannin-acyl-hydrolase (EC 3.1.1.20), is an extracellular and inducible enzyme, which catalyses the hydrolysis of some tannins and gallic acid esters. Tannins are defined as naturally occurring water-soluble polyphenols of varying molecular weight, which differ from most other natural phenolic compounds in their ability to precipitate proteins from solutions (Lekha and Lonsane, 1997). Based on the structures and properties, they are distributed in four groups: condensed tannins, gallotannins, ellagitannins and complex tannins (Khanbabaee and van Ree, 2001). Condensed tannins, also known as polymeric proanthocyanidins, are composed of flavonoid units (Bhat et al., 1998). Gallotannins are esters of gallic acid linked to a sugar core, usually glucose, whereas ellagitannins are esters of ellagic acid also linked to a sugar core (Aguilar et al., 2007). Complex tannins are mixed compounds of flavonoid units linked to gallic acids by ester bonds.

Microorganisms are the main source for industrial enzymes due to their biochemical diversities, technical and financial advances. Tannase is usually obtained from bacteria, some yeasts (Aoki et al., 1976) and filamentous fungi, mainly from the following species: Aspergillus, Penicillium, Fusarium and Thichoderma (Aguilar et al., 2007). This enzyme is applied commercially in the industries of drinks and foods, chemical-pharmaceutics, brewing, and animal feed (Belmares et al., 2004). The use of tannase in large scale, however, is much limited due to a variety of factors including production cost and insufficient knowledge of the enzyme (Aguilar and Gutiérrez-Sánchez, 2001). Although the gallic acid production in a solid state culture has been evaluated (Kar et al., 1999; Kar and Banerjee, 2000; Aguilar et al., 2002), the influence of the physical nature of a matrix used in a solid culture to produce tannase and gallic acid remains unclear. In this study, we investigate the effects of polyurethane matrices in a solid state culture on Aspergillus niger-induced tannase and gallic acid production.

The biomass/substrate yield, Y_{X /S}, is calculated by the next equation:

The specific substrate uptake rate, q _S, is defined as follows:

Tannase/biomass yield, Y_{E /X}, is estimated from the linear correlation between tannase activities, E (U/L) and biomass concentration, X (g/L). The yield coefficient is defined as Y_{E /X} (units of tannase per gram X).

The specific rate of products, q _P, is defined as follows:

The biomass production of Aspergillus niger Aa-20 reached the highest value (1.30 g/L) in the semi-discontinuous matrix, whereas discontinuous and continuous matrices gained 0.81 and 0.68 g/L, respectively (Fig.1). Table 1 presents the values of the kinetic parameters associated with the fungal growth, the specific growth rate µ and the maximum growth X _max.

Fig. 1 Growth of Aspergillus niger Aa-20 in the three different matrices of PUF: (a) semi-discontinuous; (b) continuous; (c) discontinuous. Line represents the calculated values and points are the experimental values (mean±SD)

Table 1 Kinetic parameters associated to fungal growth in the three different matrices of PUF

Table 2 shows the results of the substrate consumption. It was found that in the continuous matrix the tannic acid was consumed near completely, with only 0.30 g/L remained in the broth at the end of the culture. In the semi-discontinuous matrix, a concentration of 0.70 g/L was remained. However, in the discontinuous matrix, the substrate was not so efficiently consumed, since the remained was at 5.59 g/L.

Table 2 Kinetic parameters associated to substrate uptake in the three different matrices of PUF

Fig.2 presents the results of extracellular tannase production, and Table 3 shows the corresponding kinetic parameters. The highest enzyme activity was found at the discontinuous matrix with a 2479.59 U/L value. At the continuous and semi-discontinuous matrices, maximal activities were 543.40 and 479.39 U/L, respectively. The maximal values were obtained at a really short fermentation period around the first 12 h.

Fig. 2 Tannase activity obtained in the three different matrices of PUF

Table 3 Kinetic parameters associated to tannase production in the three different matrices of PUF

Lekha and Lonsane (1994) reported that Aspergillus niger PKL-104 produced 140 000 U/L of tannase in the solid state culture. Kar and Banerjee (2000) found high amounts of tannase produced by Rhizopus oryzae using a modified-solid fermentation. Also Aguilar et al.(2001a; 2001b) reported an 18 000 U/L of tannase as a maximum production. In these three studies, the final productions were affected by many factors including the choices of the enzyme producer microorganism and the solid support, and some culture conditions such as pH, humidity percentage, airing, etc.

Fig.3 shows that the discontinuous matrix of PUF favoured the gallic acid accumulation; 7.64 g/L was obtained during the first 12 h of the culture, whereas the continuous and semi-discontinuous matrices produced 0.74 and 0.86 g/L of gallic acid, respectively. The gallic acid accumulation was proportional to the tannase biosynthesis in the production systems (Table 4). In the discontinuous matrix supported culture system, a high accumulation of gallic acid occurred during the first 10 h, which was associated with the maximum enzymatic activity. The solid state fermentation yielded higher amounts of gallic acid during first hours of the culture, compared with a liquid fermentation (Kar and Banerjee, 2000; García-Nájera et al., 2002). In this study the gallic acid accumulation at 10 h is 1.2 times higher than the liquid fermentation (García-Nájera et al., 2002). Kar and Banerjee (2000) also reported high concentrations of gallic acid, which, however, were produced on a longer liquid fermentation.

Fig. 3 Gallic acid accumulation in the three different matrices of PUF

Table 4 Kinetic parameters associated to gallic acid accumulation in the three different matrices of PUF

The use of small particles of PUF favours the formation of fungal aggregates which exhibit a major catalytic activity, while the use of bigger particles of PUF favours the growth of longer hyphaes with a lower catalytic activity. This can be supported by the results obtained by Oostra et al.(2001) and Rahardjo et al.(2002), who demonstrated that the form of fungal cells forming the mycelium in solid state culture is extremely important because it affects the respiration and, therefore, the modulation of enzymes biosynthesis. Also, Viniegra-González and Favela-Torres (2006) indicated that the diffusion of substrates on a solid support is an important physical parameter that affects the expression of enzymes and it is seriously affected by the continuity of the solid support. In this aspect, Cerda-Montalvo et al.(2005) demonstrated the formation of substrate gradients inside the solid support using PUF in the solid state culture for tannase production. This phenomenon was originally proposed by Georgiou and Shuler (1986) and confirmed by Mitchell et al.(1991; 2004) and Olsson (1994). Strong glucose gradients were observed by Nagel et al.(2002) in solid state culture and several models have been proposed to explain these profiles of substrates in solid state culture systems by Rajagopalan and Modak (1995a; 1995b), Mitchell et al.(2004), Viniegra-González et al.(2003) and Viniegra-González and Favela-Torres (2006).

The results of the present study show that the discontinuous matrix of polyurethane is better for tannase production and gallic acid accumulation in the solid state culture bioprocess. We conclude that the physical structure of a solid support matrix has a great influence on the physiological behavior of the fungus in the solid culture than the continuous and semi-discontinuous matrices.