Christopher Augur

Advantages of fungal enzyme production in solid state over liquid fermentation systems

Abstract The present paper attempts to explain why enzyme production in solid-state fermentation (SSF) is higher than in submerged fermentation (SmF). Recent work done in our laboratory [Biotechnol. Lett. 22 (2000) 1255; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 271; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 296] related to the production of invertase, pectinases and tannases, by Aspergillus niger grown by SSF and SmF is reviewed. To do such a comparative study, logistic and Luedeking–Piret equations are used in order to estimate the values of the following coefficients: maximal specific growth rate (μM), maximal biomass level (XM), enzyme/biomass yield (YP/X) and secondary rate of production, or breakdown (k). It is shown that enzyme productivity is proportional to group, μMYP/XXM, corrected by a function of ν=k/YP/XμM. In all three cases of enzyme production studied, productivity using a SSF system was higher than in SmF. Studies with invertase resulted in higher values of μMXM. Studies with pectinases resulted in higher values of YP/XXM. Studies with tannases resulted in higher YP/X and less negative values of k. Finally, a reaction–diffusion model is presented to try to explain such differences based on micrographic measurements of mycelial aggregates for each kind of fermentation system.

Microbial tannases: advances and perspectives

In the last years, tannase has been the subject of a lot of studies due to its commercial importance and complexity as catalytic molecule. Tannases are capable of hydrolyzing complex tannins, which represent the main chemical group of natural anti-microbials occurring in the plants. The general outline of this work includes information of the substrates, the enzyme, and the applications. This review considers in its introduction the concepts and history of tannase and explores scientific and technological aspects. The “advances” trace the route from the general, molecular, catalytic, and functional information obtained under close to optimal conditions for microbial production through purification, description of the enzyme properties, and the commercial applications to the “perspectives” including expression studies, regulation, and potential uses; aspects related to the progress in our understanding of tannin biodegradation are also included.

Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid

Tannase production by Aspergillus niger Aa-20 was studied in submerged (SmF) and solid-state (SSF) fermentation systems with different tannic acid and glucose concentrations. Tannase activity and productivity were at least 2.5 times higher in SSF than in SmF. Addition of high tannic acid concentrations increased total tannase activity in SSF, while in SmF it was decreased. In SmF, total tannase activity increased from 0.57 to 1.03 IU/mL, when the initial glucose concentration increased from 6.25 to 25 g/L, but a strong catabolite repression of tannase synthesis was observed in SmF when an initial glucose concentration of 50 g/L was used. In SSF, maximal values of total tannase activity decreased from 7.79 to 2.51 IU when the initial glucose concentration was increased from 6.25 to 200 g/L. Kinetic results on tannase production indicate that low tannase activity titers in SmF could be associated to an enzyme degradation process which is not present in SSF. Tannase titers produced by A. niger Aa-20 are fermentation system-dependent, favoring SSF over SmF. Journal of Industrial Microbiology & Biotechnology (2001) 26, 296–302.

Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study

Exopectinase production by Aspergillus niger was compared in submerged fermentation (SmF) and solid-state fermentation (SSF). SSF was carried out using polyurethane foam (PUF) as the solid support. The purpose was to study the effect of sucrose addition (0 or 40 g/l) and water activity level (Aw=0.99 or 0.96) on the level of enzyme activity induced by 15 g/l of pectin. Mycelial growth, as well as extracellular protease production, was also monitored. Sucrose addition in SmF resulted in catabolite repression of exopectinase activity. However, in SSF, an enhancement of enzyme activity was observed. Protease levels were minimal in SSF experiments with sucrose and maximal in SmF without sucrose. Exopectinase yields (IU/g X) were negligible in SmF with sucrose. The high levels of exopectinase with sucrose and high Aw in SSF can be explained by a much higher level of biomass production without catabolite repression and with lower protease contamination. Journal of Industrial Microbiology & Biotechnology (2001) 26, 271–275.

Induction and repression patterns of fungal tannase in solid-state and submerged cultures

Induction and repression patterns of tannase production by Aspergillus niger Aa-20 in solid-state (SSC) and submerged culture (SmC) were established. Tannic acid and glucose were used as carbon sources. Induction and repression ratios were obtained with different concentrations of tannic acid and glucose, respectively. Tolerance to high concentrations of tannic acid by A. niger Aa-20 was lower in SmC than in SSC. In SSC an increase in tannic acid enhanced the expression of tannase activity. The addition of glucose (>20 g l−1) resulted in strong catabolite repression in SmC system. The tannase/biomass yield in SSC was at least 2 times higher than in SmC. The results presented demonstrate the capacity of SSC to minimize catabolite repression. The role of gallic acid in tannase regulation was also studied.

Microbial production of ellagic acid and biodegradation of ellagitannins

In the last years, tannin biodegradation has been the subject of a lot of studies due to its commercial importance and scientific relevance. Tannins are molecules of low biodegradation and represent the main chemical group of natural anti-microbials occurring in the plants. Among the different kinds of tannins, ellagitannins represent the group less studied manly due to their diversity and chemical complexity. The general outline of this work includes information on tannins, their classification and properties, biodegradation, ellagic acid production, and potential applications. In addition, it describes molecular, catalytic, and functional information. Special attention has been focused on the biodegradation of ellagitannins describing the possible role of microbial enzymes in the production of ellagic acid.

Culture Conditions Dictate Protease and Tannase Production in Submerged and Solid-State Cultures of Aspergillus niger Aa-20

Undesirable protease production by Aspergillus niger Aa-20 in submerged culture and solid-state culture was evaluated using different concentrations of tannic acid as sole carbon source in a model system designed for tannase production. Protease production was found to be dependent on the culture system used (submerged culture or solid-state culture) and on the initial tannic acid concentration. Expression of protease activity in submerged culture was higher (up to 10 times) than activity obtained in solid-state culture, using identical culture medium composition. In submerged culture, the lowest final protease activity (0.13 IU) was obtained with the highest tannic acid concentration, while in solid-state culture protease activity was not affected by changes in initial substrate concentration. Absence of detectable proteolytic activity in solid-state culture is related to high production of tannase enzyme. Hence, the use of solid-state culture for fungal enzyme production may allow for higher and more stable enzyme titers present in culture extracts.

Extraction and analysis of ellagic acid from novel complex sources

Ellagic acid (EA) was quantified by reversed-phase high-performance liquid chromatography (RPHPLC) coupled with photodiode array detection (DAD) in five fine-powdered plants collected from the semiarid region of Mexico. Samples analysed included Jatropha dioica branches (Dragon’s blood), Euphorbia antisyphyllitica branches (Candelilla), Turnera diffusa Willd leaves (Damiana), Flourensia cernua leaves (hojasén) and Punica granatum husk (pomegranate) at two maturity stages (“turning” or intermediate and maturated fruit, considered as positive controls). The results demonstrated high EA concentrations in all tested samples which are novel sources of this natural antioxidant. The method developed for the EA analysis is fast and it showed an excellent linearity range, repeatability, intra-and inter-day precision and accuracy with respect to the methods reported for the EA analysis.

Use of Solid State Fermentation for the Production of Fungal Biopesticides Spores for Insect Control

Numerous viruses, bacteria, nematodes and insects cause significant losses to various crops, including coffee in tropical regions (Table 1). Biological control of coffee pest consists in using natural enemies of coffee pests in order to control their population and proliferation (Dufour et al., 1999; Bustillo, 1999; Muller et al., 1999). There are over 400 species of fungi that can attack and kill specifically nematodes and insect pests (Duponnois et al., 1996; Jenkins, 1995; Schoeller and Rubner, 1994; Segers et al., 1999). The concept of using fungal pathogens to control insects is by no means new (Daoust et al., 1982; Fargues et al., 1979; Ferron, 1967). However, in the last 20 years research stimulated mainly by the resistance of insects to chemical pesticides and by enhanced public awareness of the environment has brought closer the possibility of exploiting such organisms commercially (Hokkanen and Lynch, 1995; Leij, 1992). Recent successes in exploiting nematophagous and entomophagous fungi as biopesticides demonstrated that there could be a high potential in using such microorganisms to protect coffee plants from known pests (Bustillo, 1999; Dufour et al., 1999; Villain et al., 1999).

Initial Proteome Analysis of Caffeine-Induced Proteins in Aspergillus tamarii Using Two-Dimensional Fluorescence Difference Gel Electrophoresis

Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.