Three-phasic fermentation systems for enzyme production with sugarcane bagasse in stirred tank bioreactors: Effects of operational variables and cultivation method
Introduction
Improvements in second generation (2 G) ethanol production require consideration of environmental and economic issues, including the dependence on petroleum-based fuels and the addition of value to agricultural and industrial residues. A critical factor in many biorefining approaches for 2 G ethanol production is the high cost of the enzymes required for enzymatic hydrolysis of lignocellulosic biomass, which can have a significant impact on the price of 2 G ethanol [1], [2]. The development of improved bioprocesses for enzyme production is therefore needed in order to overcome this economic limitation.
The composition of lignocellulosic material favors its use as a renewable resource for the production of biofuels and enzymes. Vegetal biomass is mainly composed of three types of polymer: cellulose (35–50%), hemicellulose (15–35%), and lignin (10–20%). The relative proportions of these substances and the interaction among them vary according to plant species, which affects the recalcitrance of the biomass and its suitability for different purposes [3], [4], [5]. When lignocellulosic materials are used as substrates for enzyme production, a pretreatment step can increase cellulose availability. Each type of pretreatment has its advantages and disadvantages. The use of steam explosion helps to remove the hemicellulose fraction from the biomass, rendering the cellulose more available to biological and enzymatic attack. This procedure is widely used for sugarcane bagasse, because no chemicals are required (except water), energy inputs are low, and the technique is compatible with existing biorefinery systems [4], [6], [7].
Many microorganisms are able to degrade lignocellulosic materials by producing enzymatic complexes containing cellulases (cellobiohydrolases, endoglucanases, and β-glucosidases) and xylanases. Other accessory enzymes are also produced that can increase the accessibility of the cellulose and enhance the hydrolysis of lignocellulosic materials. These include lytic polysaccharide monooxygenases, pectinases, laccases, manganese peroxidase, and lignin peroxidase [3], [8], [9]. The most widely studied microorganisms are the filamentous fungi Trichoderma sp. and Aspergillus sp., which are able to secrete enzymes at very high levels [10], [11].
Cellulases and hemicellulases can be produced by two conventional fermentation methods: submerged fermentation (SmF) and solid-state fermentation (SSF). The submerged fermentation method has well-developed monitoring and control systems and is relatively simple to scale up, and has therefore been widely used industrially for cellulase production. On the other hand, solid-state fermentation has the advantage of being able to provide environments similar to the natural habitats of fungi, and close interaction between the microorganism and the inducer substrate can result in higher enzyme yields [12], [13]. A new sequential fermentation (SF) method (SF) recently developed by Cunha et al. [14] combines the advantages of the solid-state and submerged cultivation techniques in a single process, and can potentially be used for cellulase production in either shake flasks or pneumatic bioreactors [15].
The bioreactors used for submerged enzyme production can be pneumatically agitated, as in bubble column bioreactors, or mechanically agitated, such as in stirred tank bioreactors (STB) [16], [17]. The STB is currently the type of bioreactor most widely used in cellulase production bioprocesses. Although commercial substrates have often been used for cellulase production, there is increasing use of lignocellulosic materials for the production of cellulases and xylanases in bioreactors [15], [18], [19].
Fungal growth under different environmental conditions can result in differences in physiology and enzyme expression, as well as in the expression of enzymes with different characteristics in terms of molecular weight, kinetic parameters, and performance [13], [20]. The influence of the lignocellulosic substrate particle size in solid-state fermentations for enzyme production has been reported recently [21], [22]. However, no research has been found concerning the effect of the solid substrate particle size when employed as inducer substrate in submerged fermentation in bioreactors.
Although there has been extensive research into enzyme production, further investigation is required of the influence of operational variables such as particle size, cultivation method, and agitation conditions in stirred tank bioreactors using sugarcane bagasse as substrate. These parameters are of key importance in the design of bioprocesses for large-scale enzyme production. In order to contribute to the development of bioprocess engineering for (hemi) cellulolytic enzymes production, the present work therefore investigates the influence of cultivation method (sequential solid-state and submerged), sugarcane bagasse pretreatment and particle size, agitation speed, and pH control on (hemi) cellulases production by Aspergillus niger in a stirred tank bioreactor.
Section snippets
Substrate
The inducer substrate used was sugarcane bagasse. The in natura untreated sugarcane bagasse (USB) was provided by Edra Ecossistemas (Ipeúna, Brazil) and the steam explosion pretreated sugarcane bagasse (PSB) was provided by CTC (Piracicaba, Brazil). Both samples originated from the same region in the State of Sao Paulo (Brazil), and the steam explosion pretreatment was conducted at 17 × 105 Pa and 205 °C for 20 min. Compositional analyses of the untreated and pretreated sugarcane bagasse samples
Results and discussion
In a first step, experiments were designed to evaluate the effects of sugarcane bagasse pretreatment, substrate particle size, and cultivation method on endoglucanase production in Erlenmeyer flask cultivations. In a second step, the effects of agitation speed and cultivation method on (hemi) cellulolytic enzymes production were evaluated in stirred tank bioreactor cultivations. The effects of biomass pretreatment and pH control were then evaluated in STB cultivations. Once the most suitable
Conclusions
Endoglucanase production in shake flasks was affected by the cultivation method, with highest production achieved using the sequential fermentation method employing pretreated sugarcane bagasse with particle sizes up to 2 mm. In cultivations using a stirred tank bioreactor, the combined effects of biomass pretreatment and agitation conditions were shown to have a strong impact on (hemi) cellulolytic enzymes production, and the advantages of the SF method and biomass pretreatment were validated.
Acknowledgments
Financial support for this work was provided by the São Paulo State Research Foundation (FAPESP, Process no. 2008/56246-0 and 2011/23807-1), the Brazilian Higher Education Foundation (CAPES), the National Council for Scientific and Technological Development (CNPq), and Embrapa. The authors would also like to thank Gabriela de Sá Azarias for technical assistance during the cultivation experiments.
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