Cellulases and most industrial enzymes are produced by submerged fermentation (SF) but solid state fermentation (SSF) is used to a lesser extent. The main advantages of SSF are low technology and high volumetric productivity, thus reduced downstream processing costs. For a long time it has been thought that the main advantages of SSF are due to water limitation of the system so that a higher product concentration is attained.

Biofilm processes are used mainly for waste water treatment but they are also considered for metabolite and enzyme production (Fiedurek, 2001; Yang et al. 2005). Although fungal biofilms are less known than bacterial biofilms, they can be used for cellulase production as it has been recently showed (Villena et al. 2001).

Both SSF and biofilm fermentation (BF) depend on surface adhesion. A new fermentation category named surface adhesion fermentation (SAF) was first proposed by Gutiérrez-Correa and Villena (2003). The concept of a biofilm presumes either a population or a community of microorganisms living attached to a surface (O’Toole et al. 2000). It should be noted that adhesion and subsequent differential gene expression to generate phenotypes distinct from those of free living organisms are two unifying processes of the biofilm concept (O’Toole et al. 2000). Filamentous fungi are naturally adapted to growth on surfaces and in these conditions they show a particular physiological behaviour which it is different to that in submerged culture; thus, they can be considered as biofilm forming organisms. Actually, once spores are adsorbed to the support they grow attached to it thus forming a film. We prefer the term biofilm fermentation instead of cell immobilization because the microbe is an active and differential entity (Gutiérrez-Correa and Villena, 2003).

The importance of the water activity (a_w) in microbial physiological processes is well recognized. It is known that a_w is a critical factor affecting the growth and metabolism of fungi and, especially in SSF, it is also considered as a fundamental parameter for mass transfer (Gervais and Molin, 2003). Despite the importance of water activity in many enzyme production systems, its role in biofilm fermentation is not explored. This paper describes the effect of ethylene glycol as a non-metabolizable water activity depressor on the lignocellulolytic enzyme production by Aspergillus niger biofilms.

Aspergillus niger ATCC 10864 was used throughout the study and was maintained on potato dextrose agar slants. Duff (1988) medium was used in all experiments at initial pH 5.5. To test the effect of a_w, the same medium was used supplemented with ethylene glycol at the following final concentrations (% v/v): 5%, 10%, 15% and 20%, which correspond to a_w of 0.976, 0.971, 0.964, 0.954, and 0.942, respectively.

For SF experiments, 30 ml of the culture medium in 125 ml flasks was inoculated with 0.9 ml spore suspension (10⁶ ml^-1) to each flask and incubated at 28ºC in a shaker bath at 175 rpm. For BF experiments, polyester cloth was used as support for biofilm formation (Villena et al. 2001). These cultures were carried out in 125 ml flasks containing 2 x 2 cm squares in 30 ml Duff medium. After inoculation with 0.9 ml spore suspension they were incubated at 28ºC in a shaker bath at 175 rpm.

The spore adhesion process was not altered by 20% ethylene glycol (a_w = 0.942) as it can be seen in Figure 1. However, both biofilm and free submerged growth were depressed at high ethylene glycol concentrations. Also, at high ethylene glycol concentration hyphal turgor is negatively affected and gummy materials are secreted (Figure 1).

Under normal water activity, SF produced more biomass (3.05 g l^-1) than BF (1.6 g l^-1) as previously found (Figure 2a, b). On the other hand, depression of water activity by ethylene glycol negatively affected the growth of both culture systems, being BF the most (1.1 g l^-1 and 0.18 g l^-1 for SF and BF, respectively).

Maximal filter paper activity (FPA), endoglucanase (ENG) and xylanase (XYL) production activities were much higher in BF (2.96, 4.7 and 4.61 IU ml^-1, respectively) than in SF (1.71, 1.34 and 2.45 IU ml^-1, respectively) (Figure 2c, d, e), which is consistent with production yields reported for most of the surface adhesion fermentation processes (Gutiérrez-Correa and Villena, 2003; Viniegra-González et al. 2003). Also, there was not a significant difference in soluble protein production between both culture systems (Figure 2f). Addition of ethylene glycol decreased maximum FPA, ENG and XYL activities in both SF (0.6, 0.7 and 1.5 IU ml^-1, respectively) and BF (1.2, 1.1 and 2.4 IU ml^-1, respectively) but it increased soluble protein production, being higher in biofilm cultures.

Biomass and lactose consumption were continuously decreased as ethylene glycol concentration was increased (Figure 3). It seems that lactose consumption was hampered by mass transfer limitations due to a decrease in solute diffusion (Gervais and Molin, 2003). Thus growth could be negatively affected due to low carbon-energy availability since the fungus had to spend more energy for membrane transport, and synthesis of compatible solutes (Ruijter et al. 2004). As a weakly chaotropic compound, ethylene glycol can freely traverse the cell membrane and it may not affect hyphal turgor at low concentrations (5% to 10%) but at higher concentrations (above 10%) it will cause a water stress with general adverse effects on cellular macromolecules.

Enzyme production related to ethylene glycol concentration by SF and BF are presented in Figure 3. All tested enzyme activities strongly decreased in both culture systems although biofilm cultures generally produced more. Although all specific activities dropped at high ethylene glycol concentration, those related to biomass had the lowest decrease. It is worth mentioning that biofilm XYL extracellular specific activity per biomass increased more than two fold (from 4.2 to 10.2 IU mg^-1 biomass) (Figure 4). The intracellular activity of all enzymes evaluated did not contribute significantly to the overall enzyme activities (Figure 4c, d) in all a_w levels tested. Although Kredics et al. (2000) found that enzymatic activities of Trichoderma harzianum and the amount of enzyme secretion (expressed as relative activity) depend on the water potential (or water activity), it is not clear whether the decrease of relative enzymatic activities is directly related to a secretion limitation. According to our results it seems possible that the low water activity affects enzyme biosynthesis rather than their secretion.

In summary, it has been found that biofilm fermentation produces higher cellulolytic enzyme yields than submerged fermentation at lower biomass yields suggesting differential gene expression mechanisms related to cell adhesion (Gutiérrez-Correa and Villena, 2003). Contrary to the findings on SSF, biofilm fermentation can better resist water stress and a differential regulation of xylanase is evident under this condition. Further work is being conducted to clarify some common molecular mechanisms involved in surface adhesion fermentation.

The authors wish to thank Dr. Robert P. Tengerdy (Colorado State University) for his helpful comments, CERTINTEX (Lima, Perú) for the use of its SEM facilities, and Mr. Gianangelo Nava (CERTINTEX) for his SEM technical assistance.

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