Three-phasic fermentation systems for enzyme production with sugarcane bagasse in stirred tank bioreactors: Effects of operational variables and cultivation method

doi:10.1016/j.bej.2015.02.004

Biochemical Engineering Journal

Volume 97, 15 May 2015, Pages 32-39

https://doi.org/10.1016/j.bej.2015.02.004 Get rights and content

Highlights

•
Operational variables strongly affect the three-phasic stirred tank bioreactor cultivations.
•
Endoglucanase and xylanase production can be increased in around 3.2 and 7.5-fold.
•
There is a combined effect of agitation speed and substrate type on enzyme production.
•
A sequential fermentation method improves cellulolytic enzymes production.
•
Smaller particles of pretreated bagasse benefit enzyme production in bioreactors.

Abstract

The high cost of enzymes is one of the main bottlenecks affecting the industrial production of cellulosic ethanol, which therefore requires the development of improved bioprocesses for the manufacture of cellulases. The present work concerns the selection of operating parameters for enzyme production in three-phasic bioreactors, using sugarcane bagasse as substrate. The parameters considered included cultivation method, substrate particle size and pretreatment, agitation speed, and pH. For both shake flask and stirred tank bioreactor (STB), a new sequential cultivation method employing steam explosion pretreated sugarcane bagasse significantly improved enzyme production, compared to conventional submerged fermentation. Larger substrate particle size provided a better support for fungal growth in shake flasks, while smaller particles resulted in greater homogeneity in stirred tank bioreactors. Maximum endoglucanase and xylanase production in the STB were 1599 ± 66 and 4212 ± 133 IU L⁻¹, respectively, under sequential cultivation using pretreated bagasse particles smaller than 0.5 mm, agitation speed of 700 rpm, and pH 5.0. The findings provide useful information concerning the influence of operational variables on (hemi) cellulases production in STB three-phasic cultivations, which should contribute to the development of bioprocesses using lignocellulosic materials in large-scale bioreactors.

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.

References (38)

J.S. Van Dyk et al.
A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes – factors affecting enzymes, conversion, and synergy
Biotechnol. Adv.
(2012)
V.B. Agbor et al.
Biomass pretreatment: fundamentals toward application
Biotechnol. Adv.
(2011)
A. Limayem et al.
Lignocellulosic biomass for bioethanol production: Current perspectives: potential issues and future prospects
Prog. Energy Combust. Sci.
(2012)
M.C.A. Wanderley et al.
Increase in ethanol production from sugarcane bagasse based on combined pretreatments and fed-batch enzymatic hydrolysis
Bioresour. Technol.
(2013)
C.E. Wyman et al.
Comparative data on effects of leading pretreatments and enzyme loadings and formulations on sugar yields from different switchgrass sources
Bioresour. Technol.
(2011)
J. Barrios-Gonzalez
Solid-state fermentation: physiology of solid medium, its molecular basis and applications
Process. Biochem.
(2012)
F.M. Cunha et al.
Sequential solid-state and submerged cultivation of Aspergillus niger on sugarcane bagasse for the production of cellulase
Bioresour. Technol.
(2012)
A. Ahamed et al.
Effect of mechanical agitation on the production of cellulases by Trichoderma reesei RUT-C30 in a draft-tube airlift bioreactor
Biochem. Eng. J.
(2010)
P.D. Delabona et al.
Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production
Bioresour. Technol.
(2012)
M. Michelin et al.
Influence of volumetric oxygen transfer coefficient (k(L) a) on xylanases batch production by Aspergillus niger van Tieghem in stirred tank and internal-loop airlift bioreactors
Biochem. Eng. J.
(2013)

I. Membrillo et al.

Effect of substrate particle size and additional nitrogen source on production of lignocellulolytic enzymes by Pleurotus ostreatus strains

Bioresour. Technol.

(2008)

C.G. Schmidt et al.

Effect of particle size and ammonium sulfate concentration on rice bran fermentation with the fungus Rhizopus oryzae

Bioresour. Technol.

(2012)

F.M. Cunha et al.

Liquefaction of sugarcane bagasse for enzyme production

Bioresour. Technol.

(2014)

M.C.C. Bustamante et al.

Comparison between average shear rates in conventional bioreactor with Rushton and Elephant ear impellers

Chem. Eng. Sci.

(2013)

Y. Sun et al.

Hydrolysis of lignocellulosic materials for ethanol production: a review

Bioresour. Technol.

(2002)

J.C. Gabelle et al.

Impact of rheology on the mass transfer coefficient during the growth phase of Trichoderma reesei in stirred bioreactors

Chem. Eng. Sci.

(2012)

M. Bizukojc et al.

A kinetic model to predict biomass content for Aspergillus niger germinating spores in the submerged culture

Process. Biochem.

(2006)

P.J. Lin et al.

Effect of volumetric power input by aeration and agitation on pellet morphology and product formation of Aspergillus niger

Biochem. Eng. J.

(2010)

M. Papagianni

Fungal morphology and metabolite production in submerged mycelial processes

Biotechnol. Adv.

(2004)

Cited by (26)

Sugarcane biorefineries: potential opportunities towards shifting from wastes to products
2021, Industrial Crops and Products
Citation Excerpt :
The processing of lignocellulosic biomass by the biochemical route is the technology considered most promising in future biorefineries, based on a pretreatment process followed by enzymatic hydrolysis and alcoholic fermentation. The integration of on-site production of enzymes has been demonstrated to be advantageous, given the better specificity of the enzymatic cocktails (Delabona et al., 2012; Cunha et al., 2017; Squinca et al., 2018; Cunha et al., 2015) and the high cost of commercial enzyme preparations (Valdivia et al., 2016; Lynd et al., 2017; Johnson, 2016). The biochemical route is of great industrial interest, due to the low formation of byproducts and mild operating conditions (Rosales-Calderon and Arantes, 2019).
Biorefining involves the sustainable processing of biomass into a spectrum of marketable food and feed ingredients, bio-based products (chemicals and materials), and bioenergy (biofuels, power, and/or heat). The processing of sugarcane is a successful example of renewable bioenergy being produced on a large industrial scale. As the world largest crop by production quantity, around 1907 millions of tons of sugarcane were globally produced last year. Currently, most of the sugarcane mills produce first generation (1 G) ethanol, sugar, and energy from the burning of bagasse. However, the use of part of the sugarcane bagasse to produce second generation (2 G) ethanol is beginning to move towards an industrial reality. The 2 G ethanol process itself also generates several side-streams, which could be further used to obtain high added-value products. Integrated 1 G-2 G biorefineries, where the whole sugarcane biomass is converted to bioenergy and a suite of bio-based products, can be an attractive investment option for a future scenario of a low-carbon economy. The purpose of this review is to gather together the most recent technologies that have been developed to enable the whole use of feedstocks, byproducts, and side-streams within the sugarcane biorefinery. Possible ways to improve the efficiency of biomass processing are discussed, such as the use of low-cost additives, together with the economic and environmental aspects related to the implementation of a sugarcane biorefinery. Potential applications of sugarcane bagasse and straw to obtain valuable products including enzymes, biofertilizers, nanocellulose and lignin-derived commodities are presented as opportunities to improve the attractiveness of this business model.
Xylooligosaccharides: prebiotic potential from agro-industrial residue, production strategies and prospects
2021, Biocatalysis and Agricultural Biotechnology
The use of agro-industrial residue in new processes has encouraged the development of biotechnological strategies to obtain biomolecules. Lignocellulosic materials are rich in xylan and can be used as an alternative and inexpensive source for xylooligosaccharide (XOS) production, contributing to the sustainable generation of products with high added-value. This article provides an overview of recent methodological strategies for XOS production, their health benefits as prebiotics, purification, and technological properties for industrial application. Classified as non-digestible, XOS are prebiotics and can be used as functional foods, promoting health benefits. The main advantages of XOS include the selective stimulation of beneficial microorganisms, the suppression of pathogenic bacteria and reduction of toxic compounds from the metabolism. XOS are produced from a wide diversity of lignocellulosic materials by chemical, physical and enzymatic methods, or by a combination of them. Physicochemical methods can generate undesirable by-products, and/or a large amount of monosaccharides, and result in a more difficult purification, while enzymatic hydrolysis allows milder process conditions because the reactions are specific and less pollutant residues are generated. Technological and nutritional approaches for XOS production which allow their use in different food and pharmaceutical products, and which stimulate the dissemination of researches in other technological areas, have also been discussed.
Cellulolytic enzymes production guided by morphology engineering
2021, Enzyme and Microbial Technology
Citation Excerpt :
A wild-type strain of the filamentous fungi Aspergillus niger F-12 was obtained from the Embrapa Food Technology collection (Rio de Janeiro, Brazil), with National Register of Biological Collections under number BRM028885. This strain has been previously characterized as a good cellulolytic and hemicellulolytic enzyme producer [8,25]. The microorganism was maintained at −18 °C and reactivated at least every 90 days by incubation in slants of potato dextrose agar medium for 5 days at 32 °C.
Endoglucanase and xylanase are critical enzymes for liquefaction and enzyme hydrolysis of high solids lignocellulosic biomass to facilitate its transport and production of desired derived products. Here is reported how combinations of different spore concentrations and pH influence microbial morphology, and how this may be used to direct expression and secretion of enzymes by Aspergillus niger. While xylanase production is not affected by A. niger morphology changes, endoglucanase production is enhanced under conditions of lower stress and by morphology that results in pellets. β-glucosidase production is enhanced under dispersed morphology, which results in up to fourfold increase of this enzyme production under the tested experimental conditions. A morphologic scale (Y) is proposed based on a form factor that considers the size and frequency of each morphology class, and that points to conditions that result in high selectivity for either endoglucanase or β-glucosidase production. An equation proposed to relate enzyme activity to morphology provides a useful tool for tuning enzyme production of A. niger, where morphology is a first indication of relative enzyme activities in a fermentation broth.
Relation between pellet fragmentation kinetics and cellulolytic enzymes production by Aspergillus niger in conventional bioreactor with different impellers
2020, Enzyme and Microbial Technology
The hydrodynamic environment in bioreactors affects the oxygen transfer rate and the shear conditions during microbial cultivations. Therefore, assessment of the effect of the hydrodynamic environment on cellular morphology can contribute to favoring the production of metabolites of interest. The aim of this work was to use image analysis in order to quantify the fragmentation of Aspergillus niger pellets in a conventional bioreactor operated using different impeller speeds, air flow rates, and impeller configurations including Rushton turbines and Elephant Ear impellers, with evaluation of the influence of the hydrodynamic environment on the production of cellulolytic enzymes. An empirical kinetic model was proposed to describe the dynamics of pellet fragmentation and quantify the shear conditions. The results showed that the agitation speed affected the dynamics of pellet fragmentation in two ways, by accelerating the damage process and by increasing the magnitude of the fragmentation. Both endoglucanase and β-glucosidase production exhibited a linear relationship with the pellet fragmentation percentage, which was directly related to the shear conditions. Interestingly, β-glucosidase production was favored under high shear conditions, while the highest endoglucanase production occurred under low shear conditions. These findings may be useful for defining suitable systems and operating conditions for the production of metabolites including enzymes in bioreactors, as well as defining conditions that favour a specific pre-determined enzyme cocktail.
The enzyme biorefinery platform for advanced biofuels production
2019, Bioresource Technology Reports
The increasing demand for energy has resulted in the exploitation of fossil fuels, which led to its depletion; therefore, this has focused our attention on the development of alternative energy sources in the advanced biofuels production. Lignocellulosic biomass is available abundantly on this earth, which is renewable and a better choice for bioethanol production as well as in terms of biorefinery concept. For bioethanol production using enzyme and the biochemical platform is considered to be a better approach as it is eco-friendly and more feasible. Around 15% to 20% of the total cost of biofuel production is of the enzyme; therefore, it has become today's requirement to produce enzyme and develop other methods or processes to enhance enzyme production. This review discusses the development in fermentation and culturing technique to improve enzyme production and the pretreated solid after of hydrothermal process as the inducer in the enzyme production for advances in biofuels.
Process optimization in a stirred tank bioreactor based on CFD-Taguchi method: A case study
2019, Journal of Cleaner Production
Citation Excerpt :
The ranges of QG and N for the experiments were 3.0–7.8 L/min and 400–700 rpm, respectively. The selections of the ranges referred to the recommendations of fermentation equipment manual and cited literature (Cunha et al., 2015). As can be seen from Fig. 5, gas holdup monotonically increased with the increase of QG and N, and the increase of QG contributed more to the increase of gas holdup than that of N in the selected ranges.
Cleaner aerobic fermentation with high efficiency is of great significance to microbial production. In an aerobic fermentation process, gas holdup and power consumption are main indexes for its performance evaluation. Based on CFD-Taguchi method, effects of control factors of impeller type, gas flow rate and agitation speed on gas holdup and power consumption were studied, by taking pullulan fermentation of Aureobasidium pullulans in a stirred tank bioreactor as a case study. Innovatively, three representative viscosity stages and their corresponding viscosities were defined according to viscosity change during fermentation, so as to quantitatively optimize the fermentation process. By range analysis and analysis of variance, effects of control factors and their significant levels on fermentation performance were obtained so as to optimize the fermentation system. All control factors were significant at low viscosity stage, and insignificant at stages of medium and high viscosity except for gas flow rate at high viscosity stage (p < 0.05). Impeller of bent blade disc turbine had the highest comprehensive evaluation index during the fermentation process, with the optimal agitation speed and gas flow rate 400 rpm and 7.8 L/min at low viscosity stage, and 400 rpm and 7.8 L/min at stages of medium and high viscosity.

View all citing articles on Scopus

View full text

Three-phasic fermentation systems for enzyme production with sugarcane bagasse in stirred tank bioreactors: Effects of operational variables and cultivation method

Highlights

Abstract

Introduction

Section snippets

Substrate

Results and discussion

Conclusions

Acknowledgments

Biotechnol. Adv.

Biotechnol. Adv.

Prog. Energy Combust. Sci.

Bioresour. Technol.

Bioresour. Technol.

Process. Biochem.

Bioresour. Technol.

Biochem. Eng. J.

Bioresour. Technol.

Biochem. Eng. J.

Bioresour. Technol.

Bioresour. Technol.

Bioresour. Technol.

Chem. Eng. Sci.

Bioresour. Technol.

Chem. Eng. Sci.

Process. Biochem.

Biochem. Eng. J.

Biotechnol. Adv.