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Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process.

Pervez S, Aman A, Iqbal S, Siddiqui NN, Ul Qader SA - BMC Biotechnol. (2014)

Bottom Line: The distillate originated after recovery of bioethanol gave 53.0% yield.An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch.The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Karachi Institute of Biotechnology & Genetic Engineering (KIBGE), University of Karachi, Karachi -75270, Pakistan. ali_kibge@yahoo.com.

ABSTRACT

Background: Cassava starch is considered as a potential source for the commercial production of bioethanol because of its availability and low market price. It can be used as a basic source to support large-scale biological production of bioethanol using microbial amylases. With the progression and advancement in enzymology, starch liquefying and saccharifying enzymes are preferred for the conversion of complex starch polymer into various valuable metabolites. These hydrolytic enzymes can selectively cleave the internal linkages of starch molecule to produce free glucose which can be utilized to produce bioethanol by microbial fermentation.

Results: In the present study, several filamentous fungi were screened for production of amylases and among them Aspergillus fumigatus KIBGE-IB33 was selected based on maximum enzyme yield. Maximum α-amylase, amyloglucosidase and glucose formation was achieved after 03 days of fermentation using cassava starch. After salt precipitation, fold purification of α-amylase and amyloglucosidase increased up to 4.1 and 4.2 times with specific activity of 9.2 kUmg⁻¹ and 393 kUmg⁻¹, respectively. Concentrated amylolytic enzyme mixture was incorporated in cassava starch slurry to give maximum glucose formation (40.0 gL⁻¹), which was further fermented using Saccharomyces cerevisiae into bioethanol with 84.0% yield. The distillate originated after recovery of bioethanol gave 53.0% yield.

Conclusion: An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch. The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production.

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Related in: MedlinePlus

Production of amylolytic enzymes and glucose formation by Aspergillus fumigatus KIBGE-IB33 at different incubation times.
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Related In: Results  -  Collection

License 1 - License 2
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Figure 3: Production of amylolytic enzymes and glucose formation by Aspergillus fumigatus KIBGE-IB33 at different incubation times.

Mentions: Optimization of various cultivation parameters plays an important role. For effective bioethanol production, fermentation time of the microbial culture and the type of the renewable carbon source used for the production of starch hydrolyzing enzymes and ethanol are among the most important factors. The aforementioned factors will ultimately direct the overall process cost for the development or the scale up of a new methodology in any bioethanol producing industry. Hence, fermentation time for the production of α-amylase and amyloglucosidase and different types of carbon sources were studied. Varieties of carbon source have been tested and can play an important role during microbial fermentation because they are the integral components for the production of cellular material and most of the time they are also associated with microbial growth [15]. Much interest has been diverted towards the utilization of economically available carbon sources in order to fulfill the industrial requirements. In the current study, to improve the production of α-amylase and amyloglucosidase for starch hydrolysis and bioethanol production, seven different carbon sources were utilized (Figure 2). The induction pattern for both amylolytic enzymes was different in various carbon sources suggesting that these hydrolases are inducible. Among all, cassava starch proved to be the most favorable inducer and contribute highest amount of enzyme units (α-amylase: 11.0 kUmg-1; amyloglucosidase: 142.0 kUmg-1) and glucose formation (81.0 gL-1) as compared to the other carbon sources tested. These results suggest that pure starch based carbon sources including sago starch, soluble starch (potato) and cassava starch are more suitable for the production of enzyme and glucose formation as compared to the different complex biomass (wheat bran and sugarcane bagasse) whereas, no enzyme production was detected when wheat starch and rice bran were used. This is because the lingocellulosic tough plant matrix was not pretreated. The cell free filtrate (CFF) collected after fermentation showed negligible titers for cellulase, pectinase and xylanase (data not shown) therefore; the starch content was not accessible for fermentation as compared to the purified starch materials. Fatima and Ali [16] tested sixteen fungal species for the production of amyloglucosidase (activity ranged between: 1.906-12.675 U ml-1 min-1) using starch in fermentation medium and the best strain they identified was A. oryzae llB-6 (12.673 ± 0.998 Uml-1 min-1). They also noticed a 30% increase in the enzyme activity when some of the process parameters were altered (pH and incubation time). Very recently, Puri et al. [17] reported the use of rice bran: wheat bran (1:1), rice bran: paddy husk (1:1) for the production of amylase and amyloglucosidase and the maximum amylase (2.72 IU) and amyloglucosidase (4.11 IU) activity was achieved when rice bran was incorporated in the fermentation medium inoculated with A. oryzae. However, A. fumigatus NTCC 1222 exhibited 341.7 U/mL amylase activity under solid state fermentation when incubated at 35°C (pH-6.0) for 06 days in nutrient salt solution [18]. In another study, detergent mediated production of glucoamylase in the presence of soluble starch is also reported using A. niger FME under shake flask system [19]. Several other researchers have also used cassava starch and cassava pulp as alternative carbon source for bioethanol production using α-amylase and amyloglucosidase [20-23]. The compositional analysis of cassava starch used in this study is presented in Table 1. The type and source of starch based materials plays a crucial role for achieving maximum bioethanol yield. The starch content in variety of biomaterials will govern the cost of bioethanol production. Therefore, before considering the production of bioethanol using a specific source, the nature of the starch molecule (linkage, granule size and shape) and the method of extraction employed must be kept in consideration. However, the values of compositional analysis of starch cannot be compared with other starch sources because of the variation of the plant source and the methods used to analyze the structure or content of starch. After selection of a suitable carbon source, fermentation time for the production of amylolytic enzymes was also studied by incubating A. fumigatus KIBGE-IB33 for different time interval ranging from 02 to 07 days. It was observed that production of α-amylase and amyloglucosidase started after 02 days of incubation and both the hydrolases were continuously produced up to day 06 and 07, respectively with a maximum titter secreted at day 03 (Figure 3). Afterwards, it was also noticed that as incubation time increases, amylolytic activity as well as glucose formation decreases. This might be due to the fact, with the passage of time the nutrients become depleted and other secondary metabolites are formed which eventually alters the pH of the medium and inhibits both the growth of the fungi as well as enzyme secretion [24]. Most of the time, secondary metabolites have a catabolic repression effect. Amylase from fungal sources is normally produced after 03 to 07 days of incubation but in some cases, enzyme secretion can be extended up to 15 days [25-28]. Prolong incubation time is one of the drawbacks of using filamentous fungi at industrial scale level which eventually increases process cost.


Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process.

Pervez S, Aman A, Iqbal S, Siddiqui NN, Ul Qader SA - BMC Biotechnol. (2014)

Production of amylolytic enzymes and glucose formation by Aspergillus fumigatus KIBGE-IB33 at different incubation times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4048251&req=5

Figure 3: Production of amylolytic enzymes and glucose formation by Aspergillus fumigatus KIBGE-IB33 at different incubation times.
Mentions: Optimization of various cultivation parameters plays an important role. For effective bioethanol production, fermentation time of the microbial culture and the type of the renewable carbon source used for the production of starch hydrolyzing enzymes and ethanol are among the most important factors. The aforementioned factors will ultimately direct the overall process cost for the development or the scale up of a new methodology in any bioethanol producing industry. Hence, fermentation time for the production of α-amylase and amyloglucosidase and different types of carbon sources were studied. Varieties of carbon source have been tested and can play an important role during microbial fermentation because they are the integral components for the production of cellular material and most of the time they are also associated with microbial growth [15]. Much interest has been diverted towards the utilization of economically available carbon sources in order to fulfill the industrial requirements. In the current study, to improve the production of α-amylase and amyloglucosidase for starch hydrolysis and bioethanol production, seven different carbon sources were utilized (Figure 2). The induction pattern for both amylolytic enzymes was different in various carbon sources suggesting that these hydrolases are inducible. Among all, cassava starch proved to be the most favorable inducer and contribute highest amount of enzyme units (α-amylase: 11.0 kUmg-1; amyloglucosidase: 142.0 kUmg-1) and glucose formation (81.0 gL-1) as compared to the other carbon sources tested. These results suggest that pure starch based carbon sources including sago starch, soluble starch (potato) and cassava starch are more suitable for the production of enzyme and glucose formation as compared to the different complex biomass (wheat bran and sugarcane bagasse) whereas, no enzyme production was detected when wheat starch and rice bran were used. This is because the lingocellulosic tough plant matrix was not pretreated. The cell free filtrate (CFF) collected after fermentation showed negligible titers for cellulase, pectinase and xylanase (data not shown) therefore; the starch content was not accessible for fermentation as compared to the purified starch materials. Fatima and Ali [16] tested sixteen fungal species for the production of amyloglucosidase (activity ranged between: 1.906-12.675 U ml-1 min-1) using starch in fermentation medium and the best strain they identified was A. oryzae llB-6 (12.673 ± 0.998 Uml-1 min-1). They also noticed a 30% increase in the enzyme activity when some of the process parameters were altered (pH and incubation time). Very recently, Puri et al. [17] reported the use of rice bran: wheat bran (1:1), rice bran: paddy husk (1:1) for the production of amylase and amyloglucosidase and the maximum amylase (2.72 IU) and amyloglucosidase (4.11 IU) activity was achieved when rice bran was incorporated in the fermentation medium inoculated with A. oryzae. However, A. fumigatus NTCC 1222 exhibited 341.7 U/mL amylase activity under solid state fermentation when incubated at 35°C (pH-6.0) for 06 days in nutrient salt solution [18]. In another study, detergent mediated production of glucoamylase in the presence of soluble starch is also reported using A. niger FME under shake flask system [19]. Several other researchers have also used cassava starch and cassava pulp as alternative carbon source for bioethanol production using α-amylase and amyloglucosidase [20-23]. The compositional analysis of cassava starch used in this study is presented in Table 1. The type and source of starch based materials plays a crucial role for achieving maximum bioethanol yield. The starch content in variety of biomaterials will govern the cost of bioethanol production. Therefore, before considering the production of bioethanol using a specific source, the nature of the starch molecule (linkage, granule size and shape) and the method of extraction employed must be kept in consideration. However, the values of compositional analysis of starch cannot be compared with other starch sources because of the variation of the plant source and the methods used to analyze the structure or content of starch. After selection of a suitable carbon source, fermentation time for the production of amylolytic enzymes was also studied by incubating A. fumigatus KIBGE-IB33 for different time interval ranging from 02 to 07 days. It was observed that production of α-amylase and amyloglucosidase started after 02 days of incubation and both the hydrolases were continuously produced up to day 06 and 07, respectively with a maximum titter secreted at day 03 (Figure 3). Afterwards, it was also noticed that as incubation time increases, amylolytic activity as well as glucose formation decreases. This might be due to the fact, with the passage of time the nutrients become depleted and other secondary metabolites are formed which eventually alters the pH of the medium and inhibits both the growth of the fungi as well as enzyme secretion [24]. Most of the time, secondary metabolites have a catabolic repression effect. Amylase from fungal sources is normally produced after 03 to 07 days of incubation but in some cases, enzyme secretion can be extended up to 15 days [25-28]. Prolong incubation time is one of the drawbacks of using filamentous fungi at industrial scale level which eventually increases process cost.

Bottom Line: The distillate originated after recovery of bioethanol gave 53.0% yield.An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch.The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Karachi Institute of Biotechnology & Genetic Engineering (KIBGE), University of Karachi, Karachi -75270, Pakistan. ali_kibge@yahoo.com.

ABSTRACT

Background: Cassava starch is considered as a potential source for the commercial production of bioethanol because of its availability and low market price. It can be used as a basic source to support large-scale biological production of bioethanol using microbial amylases. With the progression and advancement in enzymology, starch liquefying and saccharifying enzymes are preferred for the conversion of complex starch polymer into various valuable metabolites. These hydrolytic enzymes can selectively cleave the internal linkages of starch molecule to produce free glucose which can be utilized to produce bioethanol by microbial fermentation.

Results: In the present study, several filamentous fungi were screened for production of amylases and among them Aspergillus fumigatus KIBGE-IB33 was selected based on maximum enzyme yield. Maximum α-amylase, amyloglucosidase and glucose formation was achieved after 03 days of fermentation using cassava starch. After salt precipitation, fold purification of α-amylase and amyloglucosidase increased up to 4.1 and 4.2 times with specific activity of 9.2 kUmg⁻¹ and 393 kUmg⁻¹, respectively. Concentrated amylolytic enzyme mixture was incorporated in cassava starch slurry to give maximum glucose formation (40.0 gL⁻¹), which was further fermented using Saccharomyces cerevisiae into bioethanol with 84.0% yield. The distillate originated after recovery of bioethanol gave 53.0% yield.

Conclusion: An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch. The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production.

Show MeSH
Related in: MedlinePlus