Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process
© Pervez et al.; licensee BioMed Central Ltd. 2014
Received: 13 January 2014
Accepted: 20 May 2014
Published: 29 May 2014
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.
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.
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.
KeywordsAmylases Aspergillus fumigatus Biofuel Saccharification Saccharomyces cerevisiae Starch
Emerging environmental issues raised due to combustion of petroleum-based fossil fuel and emission of toxic gases have diverted the attention of scientists and researchers towards the utilization of various renewable resources for the production of bioethanol. In addition to these global concerns, other important factors that have been kept in preference are the mounting prices of the fuels and the current political scenario among the oil producing nations. Bioprocessing of renewable resources available in a particular region can help in resolving these issues. Various renewable resources in terms of agricultural biomass have been investigated for the production of bioethanol and this development proved beneficent for the biotechnological industries. Amongst various starchy materials available throughout the world; corn, sugarcane, wheat, potato , corn stover [2, 3], molasses  and purified starch  have been successfully utilized for the commercial production of bioethanol. As the demand and the cost of these starchy crop materials is increasing day by day, it has become indispensible to use substitute raw resources.
Cassava is a tropical root crop which is an economically available fermentable source and is produced by numerous countries . It is incorporated into animal feed (20.0%) and about similar proportion is converted into starch for industrial purposes whereas; some of the portion is also used as food source in several developing countries. About 50.0 to 70.0% starch content is recovered from the cassava root and due to the low ash content and rich organic nature it can be used as an ideal substrate for bioethanol production [7–9]. In addition, it can also be easily hydrolyzed by various techniques. As cassava starch does not have much industrial application in food industries as compared to corn starch, therefore it also lacks competition in terms of price and is available throughout the year due to its flexibility in terms of planting and harvesting [7, 10, 11].
In recent years, bioprocessing of various value-added products using microbial factories have been potentially explored with reference to extracellular enzymes. Agricultural biomass used as a substrate for the production of bioethanol has several limitations including high fiber content which requires high temperature for hydrolysis and this energy intensive procedure also does not provide desired yields of fermentable sugars. Hydrolysis of lingocellulosic mass by other expensive pre-treatment techniques is also time consuming. Industries also have concerns regarding the availability of the biomass throughout the year and most of the time its storage in bulk quantities is not possible due to space shortage. The development of an ideal pre-treatment method for hydrolyzing poly-phenolic lignin in the feedstock is expensive with several aforementioned limitations thus, enzymatic treatment is more preferable. Conventional method used for the production of bioethanol from cassava starch usually requires the basic gelatinization step followed by liquefaction and saccharification. The sugar formed during these processes is further fermented using either yeast or bacteria. Since, starch derived from any plant source is a complex molecule, it require various hydrolytic enzymes for its conversion into simple fermentable sugars. Among many extracellular hydrolases available, microbial amylases are frequently used for its conversion. For commercial production of amylases Aspergillus and Rhizopus species are considered most significant sources because the enzymes from these sources are generally thermostable and are available in excessive quantities [12–14].
Despite several advantages of simultaneous saccharification and fermentation using multiple organisms, there are also few shortcomings. In the initial steps, the amylolytic enzymes are produced using fungi and the starch present in the medium is allowed to hydrolyze into simpler sugars and afterwards another microbial factory (yeast or bacteria) is incorporated in the same fermentation flask to produce ethanol. In this case the primary organism (specifically fungal specie) along with amylolytic enzymes also excretes other toxic substances and proteases which in result inhibit the growth and performance of the second ethanol-producing microorganism. Along with this, the establishment of appropriate temperature for starch hydrolysis, enzymatic activity and ethanol production also plays an important role. Current research deals with the production of bioethanol from hydrolysis of an inexpensive renewable resource known as cassava starch, which is commonly available in Pakistan. The methodology used for the production of ethanol was based on double fermentation technique using partially purified fungal amylolytic enzymes for the liquefaction and saccharification of this starchy material. Keeping all disadvantages in view, this study was designed in two separate steps. In first step, amylolytic enzymes (α-amylase and amyloglucosidase) were produced using indigenously isolated filamentous fungi and were partially purified to hydrolyze cassava starch into simple fermentable sugars. In the next step, the sugar cocktail was fermented using S. cerevisiae to acquire maximum bioethanol yield.
Results and discussion
In the present study, several different fungal isolates with amylolytic activities were purified from different soil samples and preliminary identification was based on microbiological studies including cultural and microscopic characterization followed by 18S rDNA sequence analysis. Colonial and microscopic characteristics indicate that all isolates belong to genera Aspergillus. Microscopic morphology of A. fumigatus KIBGE-IB33 showed columnar and uniseriate conidial heads while, conidiophores are short and smooth. On the other hand, A. niger KIBGE-IB36 showed large, globose, dark brown conidial heads with hyaline and smooth conidiophores. Likewise, conidial heads of A. flavus KIBGE-IB34 are radiate and biseriate whereas, conidiophores are hyaline and coarsely roughened. A. terreus KIBGE-IB35 has biseriate and globose conidia with hyaline and smooth conidiophores. A. versicolor KIBGE-IB37 showed centrally rising, velvety floccose and slightly blue-green color colony on PDA with conidiophores borne from surface or aerial hyphae.
Compositional analysis of commercially available cassava starch
Cassava content (%, w/ w)
85.2 ± 4.26
1.4 ± 0.07
6.2 ± 0.31
0.9 ± 0.04
10.7 ± 0.53
89.3 ± 4.46
Purification profile of starch hydrolyzing enzymes produced from Aspergillus fumigatus KIBGE-IB33
Total volume (ml)
Total enzyme units (kU)
Total protein (mg)
Specific activity (kU mg-1)
Optimized conditions for starch hydrolysis in the presence of crude and partially purified amylolytic enzymes
Enzyme Units (kU mg-1)
Alpha Amylase (Crude)
Alpha Amylase (Partially Purified)
Amyloglucosidase (Partially Purified)
Production of bioethanol using Saccharomyces cerevisiae
Incubation time (Hour)
Glucose concentration (gL-1)
Theoretical yield (g)
Actual yield (g)
Several techniques including direct fermentation, simultaneous saccharification, simultaneous non-thermal saccharification, ultrasound assisted treatment and solid-state fermentation have been studied previously using different starchy materials and microbial sources for the production of bioethanol [20, 21, 51–56]. Along with this ethanol has also been produced by repeated batch culture through immobilization of S. cerevisiae and S. pastorianus IFO0751 on calcium alginate and porous cellulose carriers, respectively [57, 58]. Nikolic et al.  used ultrasound-assisted treatment for direct conversion of corn meal into bioethanol but the cost related to this method in amount of energy consumption is very high. Beside this, the pretreatment of multiple biomass or starch flour will also add extra budget that will eventually affect the feasibility of the bioethanol. The attempt made in the current study by consuming commercially available cheap cassava starch along with saccharification by synergistic effect of fungal amylolytic enzymes had revealed that this two-step based method can be used to achieve higher yields of bioethanol. Further, the process cost can also be reduced by using other inexpensive starchy materials or by establishing pilot programs that will scrutinize the actual feasibility and sustainability of the overall process developed.
In conclusion, an improved and effective enzymatic saccharification of inexpensive cassava starch using amylolytic enzymes from A. fumigatus KIBGE-IB33 was developed. The glucose obtained after enzymatic degradation was utilized for the bioethanol production using S. cerevisiae. Dual systematic enzyme conversion has advantages in terms of reduced energy consumption as well as increased production of fermentable sugar to achieve maximum bioethanol yield as compared to other processes. In addition, the process developed is more rapid as compared to the previously conducted studies using liquefaction and saccharification of cassava starch.
All reagents were of analytical grade and were obtained from commercial sources. Sago and cassava starch were purchased from local market, Karachi, Pakistan. Peptone (Oxoid, England), yeast extract (Oxoid, England), ammonium sulfate (Serva, Germany) and dipotassium hydrogen phosphate (Serva, Germany) were purchased from a local vendor. Whereas, magnesium sulfate, sodium hydroxide, sodium carbonate, copper sulfate, sodium potassium tartarate, sulfuric acid and potassium dichromate were acquired from Scharlau (Spain). Other chemical used including 3′, 5′- dinitrosalicylic acid (DNS) was purchased from BDH Chemicals (USA) and anthrone from MP Biomedicals (France) while, soluble starch and folin ciocalteu reagent were purchased from Merck (Germany).
Isolation and identification of filamentous fungi
The natural fungal isolates used in the current study were isolated from different soil samples that were collected aseptically from diverse vegetative fields located in Karachi, Pakistan. All the isolates were obtained after serial platting on potato dextrose agar (PDA) at 30°for 05 days according to the standard protocols. PDA medium consist of (gL-1): Boiled potato extract, 300.0 ml; dextrose, 20.0 g and agar, 16.0 g. Initially, 07 different fungal species were isolated from different samples. Among them 05 filamentous fungi were selected and characterized based on colonial morphology, 18S rDNA sequence cataloging and microscopic analysis using lactophenol blue staining method [59, 60]. After 18S rDNA gene analysis and sequencing, the sequences were analyzed by similarity search using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and were submitted to NCBI GenBank database. The confirmed sequences received the following GenBank accession numbers: KF905648, KF905649, KF905650, KF905651, and KF905652 for A. fumigatus KIBGE-IB33, A. flavus KIBGE-IB34, A. terreus KIBGE-IB35, A. niger KIBGE-IB36 and A. versicolor KIBGE-IB37, respectively. All of these isolates were tested for the amylolytic enzyme production based on starch-iodine plate method. All fungal isolates were plated on starch medium plates containing (gL-1): cassava starch, 10.0; yeast extract, 10.0; peptone, 10.0; K HPO , 1.0; and MgSO4.7H2O, 1.0. The cultures were incubated at 30°C for 05 days. After incubation the plates were flooded with potassium-iodide solution for the detection of amylolytic activity. Isolates were selected based on clear halo-zone around the fungal growth. All isolates were preserved on PDA slants at 4°C for further analysis and were sub-cultured routinely. Purified Sacchromyces cerevisiae (baker’s yeast) was purchased from the local market, Karachi, Pakistan and was grown and maintained in YPD medium (gL-1:yeast extract,10.0; Bacto-peptone, 20.0 and glucose, 20.0).
Inoculum preparation for seed culture
Total viable spores were calculated in order to prepare fungal inoculum. For this purpose spores were transferred using sterile needle from a 05 day old fungal culture grown on PDA slant and re-suspended in 10.0 ml sterile distilled water containing 0.1% Tween-20. Each suspension was serially diluted up to 10-5 in order to make homogenous spore suspension of 106 to 108spores ml-1).
Medium used for the production of amylolytic enzymes
All the selected filamentous fungi were tested for the production of α-amylase and amyloglucosidase in the presence of starch (cassava) under batch conditions using submerged fermentation technique. Production of α-amylase, amyloglucosidase and glucose was monitored at different time intervals (02 to 07 days). Basal medium used for the production of α-amylase and amyloglucosidase consists of (gL-1): Cassava starch, 20.0; yeast extract, 10.0; peptone, 10.0; K2HPO4, 1.0; and MgSO4.7H2O, 1.0. Initial pH of the medium was adjusted at 7.0 before sterilization at 121°C for 15 minutes. Fresh seed culture (10.0 ml) was inoculated in 90.0 ml production medium and incubated at 30°C for 03 days under static and anaerobic conditions. It was then further transferred into 900.0 ml medium and incubated at 30°C up to 07 days. The fungal spores were harvested by centrifuging the fermented broth at 40248 × g for 15 minutes at 4°C. The supernatant was filtered using 0.45 μ filter under vacuum. The cell free supernatant containing the amylolytic enzymes was stored at -20°C for further analysis. All the experiments were conducted in triplicates.
Optimization of physicochemical parameters for maximum enzyme yield
For the enhanced production of amylolytic enzymes, different inducing substrates (carbon sources) and fermentation time were optimized. For this purpose seven different carbon sources including sago starch, soluble starch (potato), cassava starch, wheat starch, wheat bran, rice bran and sugarcane bagasse were used in the concentration of 20.0 gL-1. A. fumigatus KIBGE-IB33 was incubated for different time intervals ranging from 02 to 07 days at 30°C under static and anaerobic condition for the selection of optimum fermentation time. Enzyme titer in terms of specific activity and glucose formation were monitored in triplicate.
Partial purification of amylolytic enzymes
The cell free supernatant containing α-amylase and amyloglucosidase was precipitated using salt precipitation method. For this purpose, salt gradient precipitation technique was employed ranging from 20.0% to 80.0% saturation using ammonium sulfate. 20% salt was incorporated gradually in CFF with continuous stirring at 4°C and was equilibrated for 18 hours. The precipitates formed were centrifuged at 40248 × g for 10 minutes at 4°C and were dissolved in citrate buffer (50.0 mM, pH-5.0). In the next run, again the 20% salt saturation was performed using the same supernatant up to 80% and every time the precipitates were equilibrated for 18 hours at 4°C. During each saturation range, the precipitates were monitored and calculated for both enzymes unit in terms of kU mg-1 of protein. The saturation level at which both hydrolyase were precipitated out with maximum unit was selected (40%).
Enzyme assay and total protein estimation
Enzyme activity of α-amylase and amyloglucosidase was estimated using DNS  and GOD-PAP method [62, 63], respectively. One unit of α-amylase is defined as the “amount of enzyme that liberates 1.0 mM of maltose per minute under standard assay condition”. Whereas, one unit of amyloglucosidase is defined as the “amount of enzyme that liberates 1.0 mM of glucose per minute under standard assay condition”. The specific units of both amylolytic enzymes are expressed in terms of kilo units per mg of protein (kU mg-1). Total protein was calculated using Lowry’s et al.  method with bovine serum albumin as standard.
Production of bioethanol
Analytical method for bioethanol analysis
For the determination of bioethanol concentration, the distillate was analyzed using Caputi et al.  method. The fermented broth was distilled using a Heating Mantle (Barnstead-Electrothermal, Thermo Scientific) at 78°C along with a quick fit distillation apparatus equipped with a Lie-big condenser and the in-let cold water attached to a chiller. All the experiments were run independently in triplicate and the results presented are the mean of three values.
Determination of bioethanol concentration by gas chromatography (GC)
Bioethanol concentration was also verified using gas chromatography system (GC17A, Shimadzu, Japan) equipped with flame ionization detector (FID). Column used was TRB-5 (30 × 0.25 mm × 0.25 μm) with nitrogen as a carrier gas (20 cmsec-1). The temperature of the detector and the injector were kept at 200°C and 130°C, respectively. The split ratio was 100:1 and the peak area of the compound was integrated against an external standard of absolute ethanol.
Physico-chemical characteristics of cassava starch
The cassava starch used in this study was purchased from the local market in Karachi, Pakistan. For the determination of total sugar anthrone method was used  whereas, reducing sugar was detected using DNS method . Total protein was performed using Lowry’s et al.  method. Glucose content was estimated using GOD-PAP method [62, 63]. Moisture content was calculated using standard drying method at 105°C until the weight become constant. Amylose and amylopectin fractions were calculated by iodometric method as suggested previously .
Cell free filtrate
3′, 5′-dinitrosalicyclic acid
Flame ionization detector
Grams per liter
Glucose oxidase per oxidase method
- kU mg-1:
Kilo units per milligrams
Potato dextrose agar.
Authors are obliged to Dr. Asma Ansari for the identification of the natural isolates used in the current study and also gratefully acknowledge the financial support from KIBGE, University of Karachi, Karachi, Pakistan.
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