versão impressa ISSN 1516-8913
Braz. arch. biol. technol. v.48 n.spe Curitiba jun. 2005
BIOPROCESS AND BIOTECHNOLOGY
Flávera Camargo Prado; Luciana Porto de Souza Vandenberghe; Carlos Ricardo Soccol*
Departamento de Engenharia Química; Universidade Federal do Paraná; Centro Politécnico; 81531-970; Curitiba - PR - Brasil
The aim of this work was to study the relation between citric acid production and respiration of Aspergillus niger LPB 21 in solid-state fermentation of cassava bagasse. The experiments were carried out in horizontal drum bioreactor coupled with a gas chromatography system. Fermentation was conduced for 144 h with initial substrate moisture of 60% using heat-treated cassava bagasse as sole carbon source. The exhausted air from the bioreactor was analyzed for the monitoring of CO2 produced and O2 consumed in order to estimate the biomass biosynthesis by the fungal culture. The metabolic activity of A. niger growth was associated to citric acid production. The system using FERSOL software determined 4.372 g of biomass/g of consumed O2. Estimated and analytically determined biomass values followed the same pattern showing that the applied mathematical model was adapted.
Key words: Citric acid, solid-state fermentation, cassava bagasse, horizontal drum bioreactor, fungus respiration
Este estudo permitiu verificar a relação ente a produção de ácido cítrico e a respiração do Aspergillus niger LPB 21 na fermentação no estado sólido do bagaço de mandioca. Os experimentos foram realizados em biorreator tipo tambor horizontal acoplado com um sistema de cromatografia gasosa. A fermentação foi conduzida durante 144 h com 60% de umidade inicial do substrato usando bagaço de mandioca termicamente tratado como única fonte de carbono. O ar de saída do biorreator foi analisado para monitorar a produção de CO2 e o consumo de O2 com o objetivo de estimar a biomassa sintetizada pelo fungo. A atividade metabólica do crescimento do Aspergillus niger está associada à produção de ácido cítrico. Usando o software FERSOL, o sistema determinou uma biomassa de 4.372 g de biomassa/g de O2 consumido. Os valores da biomassa estimada e da determinada analiticamente seguiram a mesma tendência mostrando que o modelo matemático aplicado foi adaptado.
Among the organic acids industrially produced, citric acid is the most important in quantitative terms with an estimated annual production of about 1000,000 tons (Soccol et al., 2003). The annual growth of its demand/consumption rate is around 3.5 -4.0% (Pandey et al., 2001; Vandenberghe et al., 2000a). The food industry consumes about 70% of total citric acid produced and pharmaceutical industries consume about 12%, and the remaining 18% are consumed by other industries (Soccol et al., 2003; Pandey et al., 2001).
Almost entire quantity of citric acid is obtained through submerged fermentation of starch or sucrose based media, using the filamentous fungus Aspergillus niger (Vandenberghe et al., 2000b). Solid-state fermentation (SSF) has been an alternative method for citric acid production using agro-industrial residues (Prado, 2002). It offers potential advantages in the treatment and value addition of these residues (Brand et al., 2002). SSF is characterized by the development of microorganisms in a low-water activity environment on a non-soluble material acting both as nutrient source and physical support (Pandey et al., 2000; Soccol, 1994).
In recent years, a considerable interest has been shown in using agricultural products and their residues as alternative sources of carbon such for citric acid production by A. niger (Soccol, 2001; Kolicheski, 1995; Soccol et al., 1999; Vandenberghe et al., 2000c). A variety of agro-industrial residues and by-products have been investigated with SSF techniques for their potential to be used as substrates for citric acid production (Vandenberghe et al., 2000a). A cost reduction on citric acid production can be achieved by using less expensive substrates, such as apple pomace, carrot and orange waste, cassava bagasse, coffee husk, corncob, kiwifruit peel, mussel processing wastes, okara (soy residue), rice and wheat bran (Vandenberghe et al., 1999; Garg and Hang, 1995; Aravantinos-Zafiris et al., 1994; Khare et al., 1995; Hang and Woodams, 1998; Vandenberghe, 2000). These residues are very well adapted to solid-state cultures due to their cellulosic and starchy nature. There has been an increasing trend towards efficient utilization of these residues, besides being a form of reducing environmental concerns (Soccol and Vandenberghe, 2003).
Brazil is the second largest producer of cassava, contributing with 23 million tons/year (Lu et al., 1997). Industries that produce starch from cassava root generate daily thousands of tons of cassava bagasse (CB), a solid residue generated in the starch extraction process during separation stage (Vandenberghe et al., 2000a; Pandey et al., 2000; Vandenberghe et al., 2000c). It is composed basically of fibers and residual starch that is not extractable. CB is disposed in the environment causing serious pollution problems due to its high organic material content and biodegradability (Pandey et al., 2000). Utilization of CB by microbial fermentation has been shown to be promising.
The aim of this work was to evaluate the relationship between citric acid production by SSF of cassava bagasse and the respiration of Aspergillus niger LPB 21. SSF was employed using an horizontal drum bioreactor (semi-pilot scale) that was coupled with a gas chromatography system to evaluate the release of CO2 and the O2 production.
MATERIALS AND METHODS
A strain of Aspergillus niger LPB 21 was grown on potato-dextrose-agar (PDA) medium. The slants were incubated at 28 ºC for seven days and preserved at 4 ºC for two months.
Substrate and Its Thermal Treatment
Cassava bagasse (CB) was obtained from Agroindustrial Paranaense de Polvilho Ltda. (Paranavaí - PR, Brazil). It was ground in a mill and was sieved to obtain fractions between 0.8 -2.0 mm particle size. Heat treatment was carried out in order to gelatinize the residual starch of CB, by adding 110 ml distilled water for 100 g of dry CB. Samples were heated at 121 ºC for 20 minutes (Vandenberghe, 2000).
Treated CB was supplemented with a salt solution containing (g/l): urea 2.93, KH2PO4 1.86 and FeSO4.7H2O 0.0105. The solution was sterilized at 121 ºC for 15 min. After cooling, methanol (4% v/v) was added under sterile conditions.
Spores of A. niger were produced in 250 ml Erlenmeyer flasks containing 40 ml PDA medium at 28 °C for seven days. The spores were harvested by homogenization with a solution 0.01% of Tween 80 and glass beads. The obtained suspension containing about 108 spores/ml was stored at 4 ºC for seven days.
Solid-State Fermentation (SSF)
SSF was conducted in horizontal drum bioreactor (semi-pilot scale) with 2 kg of dry substrate. Treated and inoculated substrate was placed inside the fermenter. As shown in Fig. 1, the horizontal drum bioreactor consisted of a shovel coupled to a motor axis that rotated with a controlled speed. Thus, material was revolved 3 to 4 times a day. After 20 h of fermentation, saturated air was inserted continually into drum (360 dm3/h) in order to control substrate temperature and moisture (Vandenberghe, 2000). Fermentation was carried out for 144 h at room temperature with initial substrate moisture of 60%.
Kinetic Study of Citric Acid Production
A kinetic study was carried out in order to verify the evolution of some important factors during fermentation. Every 24 h, samples were collected for evaluation of citric acid production as well as the changes of pH, protein, residual starch and reducing sugars. All determinations were conducted in triplicate.
Estimation of Growth
The respiratory metabolism of the microorganism was evaluated by determining the O2 consumption and CO2 production. This was utilized to estimate the biomass biosynthesis by the fungal culture. The exhausted saturated air from the bioreactor was passed through silica gel column and then analyzed by gas chromatography in order to determine the oxygen uptake rate and the CO2 evolved during the process.
Citric acid extraction was carried out by mixing 5 g fermented sample with 50 ml of distilled water on a magnetic stirrer for 10 min and filtering through filter paper. The filtrate obtained was subjected to high performance liquid chromatograph (HPLC) analysis using a Shimadzu LC-10AD.
A temperature of 60 ºC and 5 mM H2SO4 and the mobile phase at a flow-rate of 0.6 ml/min were used. Citric acid was detected in the column eluate by differential refractometer (Shimadzu RID-10A). Protein determination was carried out following Stutzer method (Vervack, 1973), reducing sugars by the Somogyi-Nelson method (Somogyi, 1945; Nelson, 1944) and residual starch through enzymatic method proposed by "National Starch Chemical Corporation" (1985). Biomass analytical determination was made subtracting the quantity of protein in a certain time of initial quantity of protein present in the substrate.
RESULTS AND DISCUSSION
One of the most important factors of SSF with filamentous fungus, both in laboratory and industrial scale is the estimation of biomass. Methods used in liquid fermentation cannot be applied in SSF. This fact is due to the intense adhesion of filamentous fungus mycelium to the solid substrate/support and to the SSF system heterogeneity. O2 consumption and CO2 production is the result of metabolic activity of microorganisms from which they obtain the necessary energy for growth and maintenance. Besides, the metabolic activity is associated to growth and it can be employed for biomass biosynthesis estimation (Raimbault et al., 1997).
Pintado et al. (1998) showed that an environment with high concentrations of CO2 has a positive effect on citric acid synthesis. In fact, low oxygen environment is directly involved with growth limitation, which is crucial for citric acid production. Low aeration rates are supposed to limit the respiration activity of A. niger and, consequently, to turn the metabolism to citric acid synthesis.
The study of citric acid production by A. niger in SSF revealed the importance of a CO2 rich atmosphere. The high partial pressure of CO2 probably retarded spores liberation of the filamentous fungus and favors citric acid synthesis (Vandenberghe, 2000). It is known that growth limitation is a very important factor in citric acid production. Consequently, it is possible to optimize citric acid accumulation by retarding the start of aeration. A different strategy of aeration, based on its retardation, was adopted for A. niger in order to control the metabolism of growth. Preliminary studies in columns (laboratory scale) showed that the start of air supply after 20 h, the concentration of citric acid reached 309 g/kg of dry cassava bagasse (DCB). However, when aeration started at 0 h, the production was only 267.3 g/kg DCB. The difference represented 13.5% (Vandenberghe, 2000). Thus, aeration in horizontal drum was started after 20 h of fermentation.
Kinetics of Citric Acid Production by SSF in Horizontal Drum Bioreactor
Citric acid production and other characteristic parameters, such as substrate consumption, pH evolution and biomass, were followed during SSF of A. niger with cassava bagasse. Fig. 2 presents the results obtained for SSF of CB in horizontal drum bioreactor. Maximal citric acid accumulation was reached in 144 h corresponding to 26.9 g/100 g of DCB. Starch consumption was 38.9 g/100 g of DCB and the total yield was 69%. These results were considered satisfactory. It is also important to point out other factors that could affect the metabolism of the fungus and citric acid production such as heat and oxygen transfer which are the main scale-up problems of SSF.
Respiratory Metabolism Analysis
A mass balance was carried out for the estimation of oxygen uptake rate (OUR) and CO2 evolved in terms of volumetric flow (L/h). If exhausted airflow (Fout) is known and the inlet airflow is Fin, the following equations could be considered:
It is known that:
Then, the equation (7) relates the inlet and the outlet airflow:
The mass balance for oxygen is given in order to evaluate the volumetric flow of uptake O2:
For the estimation of OUR and CO2 evolved in mass flow units (mol/h), it was considered that the air was an ideal gas at the respective volumetric flow (VO2uptake and VCO2out) and the proper corrections for temperature conditions.
Considering the balance of OUR, it was obtained the equation 9 (Pandey et al., 2001).
From the results of the OUR and CO2 production, some bioprocess parameters were estimated. The estimation of biomass at a certain time (Xn) consisted of assuming values for its yield based on oxygen consumption (YX/O) and biomass maintenance coefficient (mX). FERSOL software (Rodriguez-Leòn et al., 1988) was used in the calculations. Seven points of biomass were considered and analytically determined at 0, 24, 48, 72, 96, 120 and 144 h of fermentation. The software allowed the determination of the equation coefficients by successive approach. From the values of OUR and CO2 production, obtained experimentally, the system determined a biomass yield (YX/O) of 4.372 g of biomass/g of consumed O2 and a biomass maintenance coefficient (mX) of 0.0162 g of consumed O2/(g of biomass.h).
Fig. 3 presents the evolution of O2 and CO2 percentages during fermentation such as estimated biomass and analytical determined biomass. The production of CO2 did not exceed 0.4%. These results showed that the limitation of growth was excessive and probably the strategy of retarding aeration in 20 h was not favorable to this system. This was also showed by the value of biomass production during fermentation, which was only 0.87 g/100 g of DCB.
After 50 h of fermentation CO2 production reached its maximum. At this point, citric acid production was about 3 g/100 g of DCB. These facts showed that the microorganism gave preference to citric acid production and not to biomass formation. The difference between estimated and analytical determined biomass, mainly after 72 h of fermentation, was an indicative that indirect method of biomass determination through on-line monitoring of CO2 production could probably correct the errors presented in biomass determination by analytical methods.
This work showed the feasibility of using A. niger LPB 21 in SSF of cassava bagasse for citric acid production. From evolution of kinetic parameters of fermentation, such as CO2 production and O2 uptake, it was observed that low respiration rates favoured the production of high concentrations of citric acid. On-line monitoring of fermentation allowed the determination of the relationship between CO2 evolution, biomass and citric acid production by Aspergillus niger.
Citric acid production in horizontal drum bioreactor (semi-pilot scale) reached 26.9 g/100 g DCB corresponding to a yield of 69% based on starch consumption. In this case, growth was limited excessively and there was not enough biomass to produce higher levels of citric acid. Besides, other operational factors, such as temperature control, probably affected the fungus growth and citric acid synthesis.
It is very important to understand how all process parameters act on fungus metabolism and the synthesis of some metabolites. When working at larger scales, a large number of factors can affect process behavior such as oxygen and heat transfer, particle size, layer, type and shape of bioreactor. Consequently, a detailed scale-up study must be conducted in order to test every factor and their influence on a specific process.
Flávera Camargo Prado and Carlos Ricardo Soccol thank CAPES and CNPq, respectively for financial support, latter for a scholarship under the conditions of a Scientific Productivity Scheme.
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Received: September 29, 2004;
Revised: February 25, 2005;
Accepted: March 25, 2005.
* Author for correspondence