Production of lignocellulolytic
enzymes by Aspergillus
Financial support: This work was partially supported by INCAGRO (Ministry of Agriculture, Perú) and CONCYTEC (Ministry of Education, Perú) and by PhD grants to G.K.V. (INCAGRO and CONCYTEC).
Keywords: biofilm, cellulase, ethylene glycol, water activity, xylanase.
enzyme production by Aspergillus
Cellulases are increasingly used by several industries including fruit processing, feed production, textiles and others (Bhat, 2000; Kirk et al. 2002). 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 (Hölker et al. 2004). 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. An additional but less investigated advantage of SSF may be enhanced physiological processes in cell adhesion or biofilm formation that is characteristic for SSF.
Biofilm processes are used mainly for waste water treatment but they are also considered for metabolite and enzyme production (Freeman and Lilly, 1998; Fiedurek 2001; Iqbal and Saeed, 2005; Wu et al. 2005; Yang et al. 2005; Skowronek and Fiedurek, 2006). 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. Biofilms can be developed on either biotic or abiotic surfaces from a single species or as a community derived from several species (O’Toole et al. 2000; Fenchel, 2002). 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; Ghigo, 2003). 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 according to our former concept. The advantages of this form of growth have been industrially exploited by two culture systems: SSF and cell immobilization on inert surfaces.
Technology of cell immobilization was highly developed during the last two decades based on the operative advantages in the productive process instead of physiological issues (Groboillot et al. 1994). Natural adsorption on solid supports is an immobilization technique that it has been used with filamentous fungi thus neglecting its study as a way of biofilm formation. 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).
importance of the water activity (aw) in microbial physiological
processes is well recognized. It is known that aw is a
critical factor affecting the growth and metabolism of fungi (Kredics
et al. 2000; Parra et al. 2004)
and, especially in SSF it is also considered as a fundamental parameter
for mass transfer (Gervais and Molin, 2003). Likewise,
the production and secretion of enzymes could be affected by water
activity (aw) and by the nature of the aw depressor
(Acuña-Argüelles et al. 1994; Kredics
et al. 2000; 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 water activity depressor
on the lignocellulolytic enzyme production by Aspergillus
(1988) medium was used in all experiments. The culture medium
contained per liter:
To test the effect of aw, the same medium was used supplemented with ethylene glycol at the following final concentrations (% v/v): 5%, 10%, 15% and 20%.
ml of the culture medium in 125 ml flasks was inoculated with 0.9
ml spore suspension to each flask. After inoculation the flasks were
cloth 100/1 (65% denier and 35% textured polyester with circular stitch),
was used as support for biofilm formation (Villena et
al. 2001). 2 x
Water potential for each ethylene glycol concentration in the culture medium was measured using a WESCOR HR-33T Dew Point Microvoltimeter model 5103 with C-52 sample chamber. Water potential and water activity are related by ΨVm = RTln(aw), where Ψ is the water potential (Pa), Vm is the molar volume of water (mol m-3), R = 8.314 is the gas constant (J mol -1 K -1), T is the temperature (K) and aw is the water activity. Under the conditions used the water activity of the cultured medium containing 0%, 5%, 10%, 15% and 20% of ethylene glycol were 0.976 (Ψ = -3.35 MPa), 0.971 (Ψ = -4.05 MPa), 0.964 (Ψ = -5.03 MPa), 0.954 (Ψ = -6.5 MPa) and 0.942 (Ψ= -8.25 MPa), respectively.
samples were immersed in 10% glycerol for 2 hrs at
from BF was determined by removing polyester squares from the fermentation
broth at different time intervals, washing them three times by shaking
in 30 ml
intracellular activity measurement, biofilm or mycelial biomass was
washed three times with citrate buffer
Cellulase as filter paper activity (FPA), endoglucanase (ENG) and xylanase (XYL) were measured from the fermentation broth as previously reported (Dueñas et al. 1995). One international unit (IU) of enzyme activity was defined as the amount of enzyme that releases 1 µmol product per min (glucose equivalents for FPA and ENG and xylose equivalents for XYL). Soluble protein and lactose in the fermentation broth were determined by the standard Lowry and 3, 5-dinitrosalicylic acid methods, respectively.
effect of water activity on Aspergillus
time course profiles of fermentation under normal and water stress
conditions are depicted in Figure 2. Due to
the inoculation process used for biofilm cultures neither freely floating
mycelium nor sloughing were observed (Papagianni et
al. 2002; Papagianni and Mattey, 2004).
Under normal water activity, SF produced more biomass (
FPA, ENG and 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
2 c, d, e), which it is consistent with production yields reported
for most of the surface adhesion fermentation processes (Gutiérrez-Correa
and Villena, 2003; Viniegra-González et al. 2003;
Hölker et al. 2004). This difference cannot be ascribed
to the biomass generated in both systems as it was concluded by Díaz-Godínez
et al. (2001) who suggested that increases in exopectinase production
by SSF system were related to better fungal growth but not to higher
productivity of the enzyme. Also, there was not a significant difference
in soluble protein production between both culture systems (Figure
Comparison of fermentation variables at 72 hrs of fermentation between SF and biofilm cultures at different ethylene glycol levels are depicted in Figure 3. Biomass and lactose consumption were continuously decreased as ethylene glycol concentration was increased. As stated above, 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 (Hallsworth et al. 2003). Also, biomass yields (Yx/S) in both SF and BF systems decreased linearly almost at the same rate as ethylene glycol concentration increased (y = -0.0408x + 0.4675, R2 = 0.923, and y = -0.039x + 0.3839, R2 = 0.554, respectively). The low coefficient of correlation between biofilm Yx/S and ethylene glycol concentration may indicate that water stress in this type of culture is stronger than in submerged cultures, possible due to the participation of matrix potential in addition to osmotic potential in the former culture because of the presence of cloth and the biofilm structure itself (Gervais and Molin, 2003).
production related to ethylene glycol concentration by SF and BF are
presented in Figure 3. As it can be seen, all
tested enzyme activities strongly decreased in both culture systems
although biofilm cultures generally produced more. In submerged cultures
the decrease of FPA, ENG and XYL activities was linearly correlated
with the increase of water depressor concentration (R2
= 0.978, 0.831 and 0.841, respectively), indicating a clear direct
negative effect of water stress on fungal physiology (Acuña-Argüelles
et al. 1994; Díaz-Godínez et al. 2001). However,
in biofilm cultures more complex phenomena may be implicated since
the decrease of FPA, ENG and XYL activities due to increasing amounts
of ethylene glycol follows different patterns. On the other hand,
extracellular protein production increased at high water depressor
concentrations in both culture systems contrary to the results obtained
for exopectinase production in SSF and SF cultures (Acuña-Argüelles
et al. 1994; Díaz-Godínez et al. 2001). The reason
for this finding is not clear but it may be related to some type of
defence mechanism. Hallsworth et al. (2003) have
found that induced water stress resulted mostly in the upregulation
of proteins involved in stabilization of biological macromolecules
and membrane structure. Extracellular and intracellular enzyme specific
activities expressed as both IU per mg extracellular or intracellular
protein and IU per mg biomass are depicted in Figure
4. 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). It has been considered that in A.
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.
M.; GUTIÉRREZ-ROJAS, M.; VINIEGRA-GONZÁLEZ, G. and FAVELA-TORRES,
E. Effect of water activity on exo-pectinase production by Aspergillus
BHAT, M.K. Cellulases and related enzymes in Biotechnology. Biotechnology Advances, August 2000, vol. 18, no. 5, p. 355-383. [CrossRef]
G.; SORIANO-SANTOS, J.; AUGUR, C. and VINIEGRA-GONZÁLEZ, G. Exopectinases
produced by Aspergillus
DUFF, Sheldon J.B. Use of surface-immobilized Trichoderma in batch and fed-batch fermentations. Biotechnology and Bioengineering, March 1988, vol. 31, no. 4, p. 345-348. [CrossRef]
FENCHEL, Tom. Microbial behavior in a heterogeneous world. Science, November 2002, vol. 296, no. 5595, p. 1068-1071. [CrossRef]
Jan. Production of gluconic acid by immobilized in pumice stones
mycelium of Aspergillus
FRANCIS, Febe; SABU, A.; NAMPOOTHIRI, K. Madhavan; SZAKACS, George and PANDEY, Ashok. Synthesis of α-amylase by Aspergillus oryzae in solid-state fermentation. Journal of Basic Microbiology, October 2002, vol. 42, no. 5, p. 320-326. [CrossRef]
FREEMAN, Amihay and LILLY, Malcolm D. Effect of processing parameters on the feasibility and operational stability of immobilized viable microbial cells. Enzyme and Microbial Technology, October 1998, vol. 23, no. 5, p. 335-345. [CrossRef]
GERVAIS, Patrick; FASQUEL, Jean-Philippe and MOLIN, Paul. Water relations of fungal spore germination. Applied Microbiology and Biotechnology, December 1988, vol. 29, no. 6, p. 586-592. [CrossRef]
GERVAIS, Patrick and MOLIN, Paul. The role of water in solid-state fermentation. Biochemical Engineering Journal, March 2003, vol. 13, no. 2-3, p. 85-101. [CrossRef]
GHIGO, Jean-Marc. Are there biofilm-specific physiological pathways beyond a reasonable doubt? Research in Microbiology, January-February 2003, vol. 154, no. 1, p. 1-8. [CrossRef]
HALLSWORTH, John E.; HEIM, Sabina and TIMMIS, Kenneth N. Chaotropic solutes cause water stress in Pseudomonas putida. Environmental Microbiology, December 2003, vol. 5, no. 12, p. 1270-1280. [CrossRef]
HÖLKER, U.; HÖFER, M. and LENZ, J. Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Applied Microbiology and Biotechnology, April 2004, vol. 64, no. 2, p. 175-186. [CrossRef]
IQBAL, M. and SAEED, A. Novel method for cell immobilization and its application for production of organic acid. Letters in Applied Microbiology, March 2005, vol. 40, no. 3, p. 178-182. [CrossRef]
KIRK, Ole; BORCHERT, Torben Vedel and FUGLSANG, Claus Crone. Industrial enzyme applications. Current Opinion in Biotechnology, August 2002, vol. 13, no. 4, p. 345-351. [CrossRef]
O’TOOLE, George; KAPLAN, Heidi B. and KOLTER, Roberto. Biofilm formation as microbial development. Annual Review of Microbiology, October 2000, vol. 54, p. 49-79. [CrossRef]
PAPAGIANNI, M.; JOSHI, N. and MOO-YOUNG, M. Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized Aspergillus niger cultures. Journal of Industrial Microbiology and Biotechnology, November 2002, vol. 29, no. 5, p. 259-263. [CrossRef]
Maria and MATTEY, Michael. Physiological aspects of free and immobilized
Roberto; ALDRED, David; ARCHER, David B. and MAGAN, Naresh. Water
activity, solute and temperature modify growth spore production
of wild type and genetically engineered Aspergillus
RUIJTER, George J.G.; VISSER, Jaap and RINZEMA, Arjen. Polyol accumulation by Aspergillus oryzae at low water activity in solid-state fermentation. Microbiology, April 2004, vol. 150, no. 4, p. 1095-1101. [CrossRef]
M. and FIEDUREK, J. Inulinase biosynthesis using immobilized mycelium
PEIJ, Noël N.M.E.; GIELKENS, Marco M.C.; DE VRIES, Ronald P.; VISSER,
Jaap and DE GRAAFF, Leo H. The transcriptional activator XlnR regulates
both xylanolytic and endoglucanase gene expression in Aspergillus
VINIEGRA-GONZÁLEZ, Gustavo; FAVELA-TORRES, Ernesto; AGUILAR, Cristóbal Noé; ROMERO-GÓMEZ, Sergio de Jesús; DÍAZ-GODÍNEZ, Gerardo and AUGUR, Christopher. Advantages of fungal enzyme production in solid state over liquid fermentation systems. Biochemical Engineering Journal, March 2003, vol. 13, no. 2-3, p. 157-167. [CrossRef]
WU, Juan; XIAO, Ya-Zhong and YU, Han-Qing. Degradation of lignin in pulp mill wastewaters by white-rot fungi on biofilm. Bioresource Technology, August 2005, vol. 96, no. 12, p. 1357-1363. [CrossRef]
YANG, Xuehao; WANG, Bingwu; CUI, Fengnan and TAN, Tianwei. Production of lipase by repeated batch fermentation with immobilized Rhizopus arrhizus. Process Biochemistry, May 2005, vol. 40, no. 6, p. 2095-2103. [CrossRef]
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