O-STARCH-4 Two-Stage Sequential Continuous Enzymatic Hydrolysis of Cassava Starch Through In-Line Static Mixer Reactor

Transcription

1 O-STARCH-4 Two-Stage Sequential Continuous Enzymatic Hydrolysis of Cassava Starch Through In-Line Static Mixer Reactor *Saethawat Chamsart 1, 2, Areerat Theerawas 1,2, Sirichom Thungkao 1, 3, and Yaowpha Waiprib 1, 4 1 BBERG Bangsaen Biochemical Engineering Research Group: 2 Department of Biology, 3 Department of Microbiology, 4 Department of Food Science Faculty of Science, Burapha University, Chon Buri saethawa@buu.ac.th Tel Fax Abstract A state-of-the-art process with two In-Line Static Mixer Reactors (ISMR) was invented for two-stage sequential continuous enzymatic hydrolyses of cassava starch. Starch was liquefied and subsequently saccharified continuously and efficiently through the two units of ISMR. They comprised two major static dynamic mixers connected, each 0.01 m in diameter and 12.6 m in length, one for liquefaction and one for saccharification. Starch at concentrations of 40% (w/v) and 45% (w/v) was continuously liquefied by α-amyalse at 95 o C and ph 6.5 through the first unit of ISMR at feed rates of 15 and 30 ml/min, and dextrin solution from liquefaction was continuously saccharified by glucoamylase at 65 o C and ph 4.5 through the ISMR second unit at flow rates of 30 or 60 ml/min. The highest reducing sugar concentration of mg/ml with an DE of was obtained from hydrolyses of 45% (w/v) starch at a feed rate of 15 ml/min for liquefaction and a rate of 30 ml/min for saccharification because it possessed the longest residence reaction time of min from both processes. The liquefaction and subsequent saccharification have been done continuously and efficiently within ~2 h which was ~23-25 h shorter when compared to a conventional hydrolysis method using stirred tank reactor. This twostage sequential ISMR essentially showed an excellent performance with scaleable potential for the commercial production of sugars from continuous starch hydrolyses instead of batch operation. Key words:cassava starch, Liquefaction, Saccharification, Continuous starch hydrolysis,static mixer, Reactor Introduction The production of sugars from starch hydrolysis is one of the most popular methods. The production process comprises two major steps, starch liquefaction to generate sugar dextrin and sugar dextrin sacchrification to obtain mainly monosaccharide i.e. glucose in solution. The former step catalysed by alpha-amylase or by strong acid at a high temperature and the latter by glucoamylase at around 60 o C. Both steps normally are done in batch in an expensive stirred tank using a process time for both steps of about h. The first step takes ~3 h and the second step ~24 h (Srirot et al., 1982; Sawangwon, 2004) giving a range of DE from 90 to 98. Other methods for starch hydrolysis beyond using stirred tank have also been studied. For examples, Govindasamy et al. (1997) hydrolysed starch by enzymes using a twin-screw extruder and Ulibarri and Hall (1997) used hollow-fibre membrane reactor for enzymatic hydrolysis of cassava starch. However, no other novel efficient sequential 93

2 continuous method for starch hydrolyses by enzymes has been reported so far by other groups though single stage liquefaction through the ISMR (Chamsart et al., 2005a) and single stage saccharification through the same device (Chamsart et al., 2005b) have been reported. That is why our group made an attempt to invent a novel process working with the state-of-the-art ISMR for the efficient continuous liquefaction and saccharification of cassava strch with the aims to reduce as much as residence reaction time and operational process steps for the production of reducing sugar solution with an DE value of >90. Materials and methods Design and fabrication of a two-stage ISMR The two-stage sequential In-Line Static Mixer Reactor (ISMR) is composed of two major sections, the first for starch liquefaction and the second for saccharification, and each 12.6 m in length, and an addition section of 2.1-m ISMR between the two working as a heat exchanger for reducing temperature from liquefaction process suitable for subsequent saccharification. Each major section is comprised of six rows (layers) each 2.1 m in length, each seven glass tubes, 0.3 m in length with an inside and outside diameter of 10 and 12 mm. Tubes and sections were connected by 10 mm diameters T connector. Inside each glass tube is a static mixer, 24-static element Kenic type 0.24 m in length. Other components are illustrated in Figure 1. (6) (5) (2) (4) (3) (1) Figure 1. The two-stage sequential ISMR for liquefaction (2) and saccharification (6) for continuous enzymatic hydrolyses of cassava starch. Cassava flour suspension with α-amylase (1) is pumped through the ISMR first unit immersed in (2) at 95 o C. Flowing from the ISMR, sugar dextrin solution is simultaneously added by 0.01 N HCl solution (3) to decrease ph from 6.5 to 4.5 using a pump and then the mixture flows through a heat exchanger (4) made of a 2.1-m ISMR in ice to reduce temperature. Flowing from the heat exchanger, sugar dextrin solution is added by a solution of glucoamylase (5) at the same feed rate of (1) and then the mixture flows through the ISMR second unit immersed in (6) at 65 o C for complete saccharification generated here to obtain continuous reducing sugar product of the DE >98. Two-stage sequential enzymatic hydrolyses:continuous liquefaction and saccharification through ISMR This process was invented from a combination of continuous starch liquefaction and subsequent continuous saccharification through the two major units of ISMR each 12.6 m in length. Cassava starch at concentrations of 40 and 45% w/v was liquefied by a-amylase through the ISMR at flow rates of 15 and 30 ml/min with the same liquefaction above. During liquefaction and at the end of 12.6-m distance, a 0.01 N HCI solution was pumped into the ISMR using a peristaltic pump at a rate of 2.5 or 5 ml/min depending on liquefaction at the flow rate of 15 or 30 ml/min, respectively 94

3 in order to reduce the liquefaction ph from 6.5 to 4.5 and the mixture further flew through a heat exchanger of 2-m ISMR in ice to reduce its temperature from 95 o C to 65 o C optimal ph and temperature for subsequent saccharification. Flowing from the heat exchanger, and upstream before flowing through the 12.6-m ISMR saccharification unit, the mixture was continuously added by a solution of 0.8 or 0.9 % (w/v) glucoamylase (depending on the initial starch concentrations used ie. 40 or 45 % (w/v), respectively), using a peristaltic pump at the same feed rates of liquefaction ie. 15 or 30 ml/min. This gave a final concentration of 0.2% of conc glucoamylase volume per flour weight and the feed rates during saccharification through the ISMR became 30 or 60 ml/min which were two times of those liquefaction processes. The sugar samples were withdrawn from different distances of the ISMR i.e. 2.1, 4.2, 6.3, 8.4, 10.5, and 12.6 m from both liquefaction and saccharification units for further assay. Viscosity measurement The viscosity of sugar dextrin solution samples during liquefaction and of sugar solution samples during saccharification was measured by a viscometer (LvDvIII+, Brookfield, Massachusetts, USA) using a spindle sc4-18 and a cylinder 13 RP at real temperatures i.e. 95 o C for liquefaction and 65 o C for saccharificatrion. The viscosity of the samples from different conditions was measured at a shear rate range from 40 to 300 s -1 and the peak values were used to plot the curves. Reducing sugar and Dextrose Equivalent analysis The reducing sugar concentration of samples was determined using DNS assay method and using a rang of glucose concentration as a standard and the DE was determined as a fraction of the reducing sugar concentration to the concentration of total solid left in the sample solution. Results and discussion Two-stage sequential enzymatic hydrolyses: continuous liquefaction and subsequent continuous saccharification through the ISMR Cassava starch at concentrations of 40 and 45% w/v was liquefied through the ISMR at the flow rates of 15 and 30 ml/min. Flowing from the end of liquefaction section, the sugar dextrin solution was continuously added by a glucoamylase solution at the same feed rates of liquefaction of 15 and 30 ml/min giving fluid flow rates 30 and 60 ml/min during saccharification through the ISMR. This was also to dilute the sugar solution product to the required concentrations at the end of saccahrification depending on the utilisation purposes. Results in Figures 2 show that during both liquefaction and saccharification processes, in all cases of starch concentrations and feed rates, reducing sugar concentrations and DE values increased as a function of tube length due to the increase in residence reaction times. There were no significant difference of reducing sugar concentration and DE value at the end of liquefaction (12.6 m) between the two flow rates but there were difference of those values between the two starch concentrations. During subsequent saccharification process, the 95

4 reducing sugar concentrations and DE values were increasing as an increase in tube lengths, but they were ~2x lower as expected because the sugar dextrin solution from the former section was diluted ~2x by the addition of glucoamylase solution. The curves in Figures 2 reducing sugar concentration clearly plunged and the DE obviously surged between the liquefaction and the saccharification section because the glucoamylase solution was pumped into the ISMR second unit between the end of liquefaction and the beginning of saccharification. However, reducing sugars, at the end of saccharification of , , and mg/ml with the DE values of 99.29, 97.73, 83.63, and were obtained from lysis of sugar dextrin solutions (from the initial starch concentration of 45 and 40 % (w/v)) at a feed rate of 30 ml/min and from those of 45 and 40 % (w/v) at a feed rate of 60 ml/min. Statistical analysis showed significant difference of the reducing sugar concentration and DE value between the samples from the initial starch concentrations of 45 and 40 %(w/v), and between those from the flow rates of 30 and 60 ml/min. However, as they were diluted two times, the actual total product obtained would be also two volumes, thus the actual reducing sugar should be , , 210.6, and mg/unit volume. These estimated values are comparable with the values of saccharification above. Those with the DE results showed that this two-stage sequential enzymatic hydrolyses of cassava starch worked very well and could be further scaled-up for industrial use %, 15 ml/min liquefaction, 30 ml/min saccharification 45 %, 15 ml/min liquefaction, 30 ml/min saccharification 40 %, 30 ml/min liquefaction, 60 ml/min saccharification 45 %, 30 ml/min liquefaction,60 ml/min saccharification %, 15 ml/min liquefaction, 30 ml/min saccharification 45 %, 15 ml/min liquefaction, 30 ml/min saccharification 40 %, 30 ml/min liquefaction, 60 ml/min saccharification 45 %, 30 ml/min liquefaction, 60 ml/min saccharification Reducing sugar concentration (mg/ml) Liquefaction Saccharification DE Liquefaction Saccharification Distance (m) Distance (m) Figures 2. Reducing sugar concentration and DE value during continuous liquefaction and subsequent saccharification at different starch concentrations and feed rates through a state-of the-art two-stage sequential ISMR Peak viscosity They were more than 1,000 mpa s at the initial stage and down to 100 mpa s during the first 2.1 m of liquefaction as starch started to gelatinised due to just increasing temperatures of the starch solution in the ISMR. The peak viscosities were between 50 and 10 mpa s from the distances 4 to 12.6 m of liquefaction. The peak viscosities of sugar solution from saccharification during flowing through the ISMR second unit were gradually reducing as a function of tube length and lysis time with a value 96

5 between 2 and 3 mpa s which was very low because (i) the sugar dextrin solution was added with a glucoamylase solution before it flew to the ISMR second unit and this dilution clearly caused a plunging between the 12.6 m (end of liquefaction) and 15 m (start of saccharifiaction), and (ii) complete hydrolysis of sugar dextrin in the solution to obtain mainly glucose molecules (ie. known from DE value of >98). Unlike the early stage of liquefaction, the viscosity of samples after 5 m of liquefction until the end of saccharification with a value range from 30 to 2 mpa s facilitates fluid flow through the ISMR. Essentially, this is an advantage for the design and construction of a large-scale two-stage sequential ISMR for industrial use %, 15 ml/min liquefaction, 30 ml/min saccharification 45% 30 ml/min liquefaction, 60 ml/min saccharification 40% 15 ml/min liquefaction, 30 ml/min saccharification 40% 30 ml/min liquefaction, 60 ml/min saccharification Viscosity (mpa.s) Saccharification Liquefaction Distance (m) Figure 3. The peak viscosity during continuous liquefaction and subsequent saccharification at different starch concentrations and feed rates through a state-of-the-art two-stage sequential ISMR Flow regime during two-stage sequential enzymatic hydrolyses of cassava starch The fluid flow regime during enzymatic hydrolyses of 40 and 45 %w/v of cassava starch of all feed rate conditions were very laminar (Re <<980) (Figure not shown). Unlike residence reaction time, it is difficult to draw the relation between the reducing product yield and Re, whilst the residence time, controlled by the tube length and feed rate, controls the reducing sugar productivity and DE. This clearly indicates that a scale-up strategy must involve residence time as a key parameter. Conclusions The state-of-the-art process of two-stage sequential In-Line Static Mixer Reactors (ISMR) performed very well for continuous liquefaction of starch and subsequent saccarification of sugar dextrin solution for the production of sugars with the designed concentrations and DE values. The highest reducing sugar concentration of mg/ml with an DE of was obtained from hydrolyses of 45% (w/v) starch at a feed rate of 15 ml/min for liquefaction and a rate of 30 ml/min for saccharification. 97

6 From this design and operation with the desired conditions, during the feed stock is enzymatically being hydrolysed through the ISMR, the dextrin and sugar products with required concentrations and DE values can be collected from different points (reactor tube lengths) along the line. This two-stage sequential ISMR showed an excellent performance with scaleable potential for the commercial production of sugars from starch hydrolyses. Acknowledgements This is the sixth one in ten in a series of the research programme Development of Enzymatic Hydrolysis of Cassava Starch Using Thai-Base Full Cycle Biotechnology funded by the Thai National Research Council started from the year 2004 to References Chamsart, S., Sriprasit, A., Thungkal, S., and Waiprib, Y Continuous Starch Liquefaction Through In-Line Static Mixer Reactor. Proceeding of the 3 rd International Conference on Starch Technology, November. Xxx-xxx Chamsart, S., Chanon, D., Thungkao, S., and Waiprib, Y Continuous Starch Saccharification by Glucoamylase Through In-Line Static Mixer Reactor Following Liquefaction. Proceeding of the 3 rd International Conference on Starch Technology, November. Xxx-xxx Covindasamy, S., Campanella, O. H., and Oates, C.G Enzymatic hydrolysis and saccharifcation optimization of sago starch in a twin-screw extruder. Journal of Food Engineering. 3: Sawangwon, C Starch hydrolysis in a stirred tank bioreactor. Master Degree Thesis, Faculty of Science, Burapha University. Srirot, K., Rojanaritpichet, C., Piyajomkuan, K., Teea, S., and Kal, S A study of ethanol pilot plant by development of production technology from cassava root chip. Bangkok: Thailand National Research Council. Ulibarri, R.L., and Hall, G.M Saccharification of cassava flour starch in Hollow-Fibre membrane reactor. Enzyme and Microbial Technology. 21: