Abstract
Waste clay recovered from palm oil mill effluent (POME) was characterized and used as an enzyme-supporting material for the cassava saccharification process. The clay was treated by the Soxhlet extraction method to remove the residual oil and then characterized using a BET surface area analyser, XRF, XRD, FTIR, TGA, and FESEM. The chemical analysis showed that the sample had a high amount of CaO (93%) with a minor content of SiO2 (1.378%) and Al2O3 (0.707%), with a surface area of 1.15 m2/g. The XRD analysis revealed the major mineral presence to be calcite, as confirmed by FESEM analysis. The FTIR results also attested to the presence of a calcite phase and carbonate groups. To study the performance of the waste clay for enzyme immobilization application, the recovered waste clay was further used as an enzyme supporting material for enzyme immobilization in the cassava saccharification process. Results showed that the enzymes were successfully encapsulated and gave the highest immobilization yield of 70% with 2% clay concentration. In addition, the encapsulated enzymes also enhanced the reusability, where the enzyme retained 32% of its activity after seven cycles of saccharification processing.
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Characterization of Waste Clay from Palm Oil Mill Effluent and Enzyme Immobilization Study for Cassava Saccharification Process
Nurul Aini Edama,a,* Alawi Sulaiman,a Siti Noraida Abd-Rahim,a Ku Halim Ku Hamid,b and Zainuri Busu c
Waste clay recovered from palm oil mill effluent (POME) was characterized and used as an enzyme-supporting material for the cassava saccharification process. The clay was treated by the Soxhlet extraction method to remove the residual oil and then characterized using a BET surface area analyser, XRF, XRD, FTIR, TGA, and FESEM. The chemical analysis showed that the sample had a high amount of CaO (93%) with a minor content of SiO2 (1.378%) and Al2O3 (0.707%), with a surface area of 1.15 m2/g. The XRD analysis revealed the major mineral presence to be calcite, as confirmed by FESEM analysis. The FTIR results also attested to the presence of a calcite phase and carbonate groups. To study the performance of the waste clay for enzyme immobilization application, the recovered waste clay was further used as an enzyme supporting material for enzyme immobilization in the cassava saccharification process. Results showed that the enzymes were successfully encapsulated and gave the highest immobilization yield of 70% with 2% clay concentration. In addition, the encapsulated enzymes also enhanced the reusability, where the enzyme retained 32% of its activity after seven cycles of saccharification processing.
Keywords: Waste clay; BET surface area; XRD; Supporting material; Enzyme immobilization
Contact information: a: Tropical Agro-Biomass Research Group, Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA, Malaysia; b: Faculty of Chemical Engineering, Universiti Teknologi MARA, Malaysia; c: Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia; *Corresponding author: ainiedama@yahoo.com
INTRODUCTION
Enzymes (biocatalysts) have been a remarkable discovery in the field of bioprocess technology and have a range of biotechnological applications. Currently, there has been an increasing interest to enhance enzyme productivity and to increase the shelf life due to its unstable nature and sensitivity under certain conditions. In addition, using purified enzymes and discarding them after each use are costly and not economical; therefore, attempts have been made to immobilize enzymes in the bioprocessing field, especially in food and pharmaceutical technologies.
There are several advantages to immobilized enzymes, one being that they are easier to handle, which helps to prevent contamination of the substrate and enzymes in a reaction mixture. In addition, immobilized enzymes also facilitate the efficient recovery and reuse of costly enzymes with longer half-lives and less degradation (Sheldon 2007; Edama et al. 2014a).
Encapsulation is a process by which the enzymes are packaged or enclosed physically or chemically within a matrix or semi-permeable membrane layer (Abd. Rahim et al. 2013b). Through this method, the enzymes are restricted by the membrane walls (usually in a form of capsules), but free floating within the core space. The membrane itself is semi-permeable, allowing for free flow of substrates and nutrients, yet keeping the enzymes inside (Gorecka and Jastrzebska 2011); therefore, the selection of supporting material is one of the important factors that should be considered. There are several types of supporting material such as carrageenan, chitosan, and starch, but alginate is most commonly used because it has good biocompatibility, low cost, easy availability, and ease of separation; however, it has several disadvantages such as low mechanical strength and large pore size, which can cause high enzyme leakage from the alginate beads (Zhou et al. 2010; Duarte et al. 2013).
To increase the stability of the enzymes within the beads, alginate can be co-supported with an inorganic material such as clay (Edama et al.2014c). Clay is a favorable material due to its porous structure and high mechanical strength. It is also chemically inert, thermally stable, and has a low cost of production. Several studies have been conducted using fresh clay for enzymes immobilization, as exhibited in Table 1.
Table 1. Recent Studies using Fresh Clay from Different Sources for Enzyme Immobilization
One of the sources of waste clay that can be obtained abundantly is palm oil mill effluent (POME). The POME is effluent generated from the processing of a type of oil palm fruits. The final POME is the combination of different sources within palm oil mill such as condensate from the sterilization process, sludge from the clarification process, and clay bath from the hydrocyclone process.
Clay bath is a technique to separate the kernel or the shell from the oil palm fruits. The disadvantage of this technique is that it results in high waste clay generation. Direct discharge of waste clay into the current POME treatment pond may lead to serious siltation problems and cause frequent dislodging of the ponds which may increase costs.
In the present work, waste clay was used as a raw material for the enzyme immobilization study for cassava saccharification process. Saccharification refers to the process of breaking a complex carbohydrate such as starch into small units of glucose (monosaccharide) by using enzyme. The clay (enzyme supporting material) was characterized using Brunauer Emmett Teller (BET) surface area analysis, X-Ray fluorescence (XRF), X-ray diffractometry (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), and field emission scanning electron microscopy (FESEM). Then, the performance of the clay as an enzyme support material for glucose production was evaluated.
EXPERIMENTAL
Materials
The clay was obtained from clay bath palm oil mill effluent at the FELDA Serting Hilir Palm Oil Mill, Negeri Sembilan, Malaysia. All reagents and chemicals used were of analytical grade and obtained either from Sigma-Aldrich (USA) or Merck (USA).
Methods
Waste clay treatment
Soxhlet extraction using hexane as the solvent was conducted to remove the residual oil from the waste clay. First, 50 mL of fresh semi-dried waste clay was poured in a beaker and dried in an oven for 24 h. The dried clay was weighed and placed into an extraction thimble and then inserted into a 500-mL reflux flask. The extraction was carried out using 300 mL of hexane as a solvent at 150 °C. The extraction was terminated after 8 h, and the remaining hexane was removed by drying in an oven at 103 °C (Edama et al. 2014b).
Characterization techniques
Characterization of the clay was carried out to investigate the physicochemical characteristics of the material using various analytical methods. The specific surface area was measured and characterized using a Quantachrome Autosorb-1 analyzer (USA) through nitrogen adsorption at 77 K with a relative pressure (P/Po) range of 0.035 to 0.35. The chemical analysis of the clay sample was carried out by a Philips X-Ray fluorescence spectrometer (Model PW-2400, USA) and the IR spectra were obtained using a Perkin Elmer Fourier transform infrared spectrometer (Model 2000, USA).
The mineralogical composition was identified by a Rigaku Ultima III X-ray diffractometer (Japan) with a 2θ angle ranging from 15° to 75°. To obtain the differential thermal analysis (DTA)/thermogravimetric analysis (TGA) curves, a Mettler Toledo TGA/SDTA851 differential calorimeter apparatus (Switzerland) was used. The sample was heated in a silica crucible at a constant heating rate of 20 °C/min operating in a stream of N2 atmosphere with a flowrate of 40 mL/min from 35 to 1000 °C. The surface morphology of the clay sample was characterized by a Zeiss Supra 40VP FESEM (Germany) operating at an accelerating voltage to 5 kV. The sample was initially dusted on a double sided carbon tape that was placed on a metal stub and coated with a layer of gold to minimize the charging effects.
Encapsulation of enzymes
Exactly 2.5 g of waste clay (WC) was dissolved in 100 mL of a pH 6 buffer and stirred for 1 h at room temperature. Then, 2.5 g of sodium alginate was added to the clay solution and stirred for 1 h. Afterward, 2 mL of glycerol was added to the alginate-clay solution. A 2% w/v alginate-clay solution was produced by adding 3 mL of enzyme solution (1 mL of each enzyme: glucoamylase, cellulase, and alpha-amylase) into 12 mL of alginate-clay solution for a total mixture volume of 15 mL. The mixture was taken into a syringe, and beads were formed by dropping the mixture into a 0.2 M calcium chloride (CaCl2) solution. The formed beads were allowed to harden for 3 h at room temperature, dried using filter paper, and washed several times with distilled water. The filtered calcium chloride solution was collected for loading efficiency determination, which was calculated by the following equation,
(1)
where Ci is the initial protein concentration, Vi is the initial volume of enzyme solution, Cf is the protein concentration in total filtrate, and Vf is the total volume of the filtrate. The immobilization yield was defined as the yield for enzyme which was immobilized in the calcium alginate beads and expressed by the following equation,
(2)
where aimm is specific activity of immobilized enzyme (mmol/(min mg protein)) and afree is the specific activity of free enzyme (mmol/(min mg protein)).
RESULTS AND DISCUSSION
Characterization Studies
BET surface area and chemical analysis
The data in Table 2 show the BET surface area and chemical compositions of the sample. The data reveal that the waste clay had relatively low surface area (1.15 m2/g) compared to common porous materials such as activated clay (e.g., kaolinite, montmorillonite), as reported in previous studies (Muthuvel et al. 2012; Edama et al.2014c). The chemical composition of the clay sample is summarized in Table 2. The clay consists of SiO2 (1.378%), Al2O3 (0.707%), MgO (2.269%), and CaO (93.048%). The high content of CaO in the sample indicates a high amount of carbonate. This result is comparable with a study by Skels (2011), which indicated that the sample known as quicklime had a high content of CaO (95%) but low SiO2 (1.6%) and Al2O3 (0.25%) content, as well as some impurities.
Table 2. Chemical Composition and Specific Surface Area Analysis
X-ray diffraction
Figure 1 shows the XRD pattern of waste clay with a major peak at 29°, indicating that calcite is a major phase of the waste clay. It is also observed that most of the peaks correspond to h k l of 012, 104, 110, 113, 202, 024, 018, 116, 122, 1010, 208, 0012, 0210, and 1112, which demonstrated that the phase of the waste clay sample is calcite. The results were proven by comparing the h k l of the waste clay with the standard diffraction spectrum of calcite powder as reported in a previous study (Hoque et al. 2013). The strong and sharp peaks showed that the waste clay was substantially crystalline.
Fig. 1. X-ray diffraction (XRD) pattern of waste clay. C=calcite
Fourier transform infrared (FTIR) analysis
The usefulness of qualitative analysis such as FTIR from the characteristic frequencies provides information to identify the chemical compounds in the sample. The FTIR spectra of the sample were carried out in the range of 500 to 4000 cm-1, as shown in Fig. 2. The weak band at 1794 cm-1 corresponds to the C=O bonds from carbonate. Meanwhile, the peaks at 1396 cm-1 and 873 cm-1 represent the characteristic of the C-O stretching and bending modes of calcium carbonate, respectively (Witoon 2011). The sharp band observed in Fig. 2 at 712 cm-1 represents the Ca-O bonds.
Fig. 2. FTIR spectra of waste clay
Thermal behavior
The thermal analysis results of the calcite-based clay sample are exhibited in Fig. 3. The TGA pattern of the sample shows one distinct stage of weight loss, where the total weight loss of about 34% occurred from 674 to 819 °C, which is in the thermal degradation temperature range for carbonates. The results are comparable with previous studies, as summarized in Table 3. The major weight loss at 785 °C corresponding to the 34% weight loss was due to the change of the CaCO3 phase to the CaO phase (Witoon 2011). As the sample weight remained constant after 880 °C, the temperature of 900 °C was then suitable for use as the calcination temperature to ensure a complete conversion to CaO.
Fig. 3. Differential thermal and thermogravimetric analysis of waste clay sample
Table 3. Thermal Degradation Study of Waste Clay using TGA
Morphology
The morphology of the waste clay was observed from the FESEM micrograph to visualize its textural and surface structure. Figure 4 clearly shows that the waste clay had an agglomerate structure and did not have a well-defined pore structure. This result is similar to the structure of calcite-based powder as reported by Tsai (2013). This finding is consistent with the results of the BET surface area. Furthermore, the image shown in Fig. 4b depicts the morphology and crystal structure of cube-like or hexagonal calcite crystals, which were confirmed by the XRD results. This cube-like crystal makes calcite stronger more and more stable (Hoque et al. 2013). Thus, due to this stability, this material is suitable for use as a supporting material for enzyme immobilization application.
Fig. 4. FESEM micrograph of waste clay at 5000x magnification at two different spots
Enzyme Immobilization
Enzyme loading and immobilization yield
The effect of the waste clay concentration on the immobilization yield and enzyme loading for the tapioca starch saccharification process is shown in Fig. 5. It was observed that the highest yield was at 2% clay concentration with 70% activity. In general, the enzyme loadings were more than 90% for all clay concentrations and increased progressively as the clay concentration increased. The increase in loading efficiency of enzymes with high clay concentrations may be due to the formation of stronger and tighter beads; however, the immobilization yield slightly decreased as the clay concentration increased. The decrease in activity of the encapsulated enzymes in the clay beads is attributed to the diffusion limitation of the substrate to react with the enzymes (Gülay and Şanlı-Mohamed 2012). The enzymes encapsulated alginate-clay beads characterization have been thoroughly studied and published elsewhere (Edama et al. 2014b).
Fig. 5. Enzyme loading and immobilization yield at different concentrations of clay
Reusability
The reusability of the enzymes is the main advantage of the enzyme immobilization system, as it is a key factor for its cost-effective industrial use (Talekar and Chavare 2012). The reusability study of encapsulated enzymes was determined by using recovered enzymes from different cycles as proposed in a previous study (Abd Rahim et al. 2013a). The residual activity of each cycle was calculated by taking the enzyme activity of the first cycle as 100%. Figure 6 indicates the reusability patterns of the immobilized enzymes for the alginate beads and alginate-clay beads after several cycles. The encapsulated enzymes retained 65%, 56%, and 32% of their activities after the 5th, 6th, and 7th cycles, respectively. The decrease in the enzyme activities may be due to enzyme leakage resulting from the soft surface of the alginate gel. Other reasons for the leakage may be damage to the beads during the repeated use and washing steps at the end of each cycle (Gülay and Şanlı-Mohamed 2012).
Fig. 6. Reusability of alginate-clay beads in tapioca slurry hydrolysis process
CONCLUSIONS
- The major constituent of the palm oil mill effluent (POME) waste clay sample was CaO (93.048%), with minimal amounts of Fe2O3 (1.778%), SiO2 (1.378%), and Al2O3 (0.707%), as confirmed by XRD and TGA analysis. The waste clay sample also had a relatively low surface area of 1.15 m2/g.
- The FESEM analysis showed that the waste clay recovered from POME had a strong and stable structure, which made it suitable as an enzyme supporting material.
- The waste clay recovered from POME was successfully used as a supporting material in an enzyme immobilization study for glucose production. It was found that the highest yield obtained had a 2% waste clay concentration, and the encapsulated enzyme was able to be reused after up to seven cycles.
ACKNOWLEDGMENTS
This work was financially supported by University Teknologi MARA (UiTM) Cluster Grant (600-RMI/DANA 5/3/CG (16/2012) and Ministry of Higher Education (MOHE) Malaysia, Long-term Research Grant Scheme (600-RMI/LRGS 5/3 1/2012).
REFERENCES CITED
Abd Rahim, S. N., Sulaiman, A., and Ku Hamid, K. H. (2013a). “Performance of encapsulated enzymes within calcium alginate-clay beads in a stirred bioreactor for biosugar production,” Advances in Environmental Biology 7(12), 3783-3788.
Abd. Rahim, S. N., Sulaiman, A., Ku Hamid, K. H., Muhd Rodhi, M. N., Musa, M., Hamzah, F., and Edama, N. A. (2013b). “Nanoclay supporting materials for enzymes immobilization: Kinetics investigation of free and immobilized system,” Applied Mechanics and Materials 393, 115-120. DOI: 10.4028/www.scientific.net/AMM.393.115
Abd. Rahim, S. N., Sulaiman, A., Hamzah, F., Ku Hamid, K. H., Muhd Rodhi, M. N., Musa, M., and Edama, N. A. (2013c). “Enzymes encapsulation within calcium alginate-clay beads: Characterization and application for cassava slurry saccharification,” Procedia Engineering 68, 411-417. DOI: 10.1016/j.proeng.2013.12.200
Abdul Rahman, M. B., Md Tajudin, S., Hussein, M. Z, Raja Abdul Rahman, R. N. Z., Salleh, A. B., and Basri, M. (2005). “Application of natural kaolin as support for the immobilization of lipase from Candida rugosa as biocatalyst for effective esterification,” Applied Clay Science 29,111-116. DOI: 10.1016/j.clay.2014.001
Ajayi, O. A., Nok, A. J. , and Adefila, S. S. (2012). “Immobilization of cassava linamerase on Kankara kaolinite clay,” Journal of Natural Science Research 41, 1864-1874.
Duarte, J. C., Rodrigues, J. A. R., Moran, P. J. S., Valenca, G. P., and Nunhez, J. R. (2013). “Effect of immobilized cells in calcium alginate beads in alcoholic fermentation,” AMB Express 3(31), 1-8. DOI: 10.1186/2191-0855-3-31
Edama, N. A., Sulaiman, A., and Abd Rahim, S. N. (2014a). “Enzymatic saccharification of Tapioca processing wastes into biosugars through immobilization technology,” Biofuel Research Journal 1, 2-6.
Edama, N. A., Sulaiman, A., Ku Hamid, K. H., Abd Rahim, S. N., Baharuddin, A. S., and Mokhtar, M. N. (2014b). “Encapsulation of multi-enzymes on waste clay material: Preparation,characterization and application for tapioca starch hydrolysis,” Applied Mechanics and Materials 548-549, 77-82. DOI: 10.4028/www.scientific.net/AMM.548-549.77
Edama, N. A., Sulaiman, A., Ku Hamid, K. H., Muhd Rodhi, M. N., Musa, M., and Abd Rahim, S. N. (2014c). “Physico-chemical and morphological changes of sayong kaolinite clay treated with sulphuric acid for enzyme immobilization,” Advanced Material Research 832, 589-595. DOI: 10.4028/www.scientific.net/AMR.832.589
Gorecka, E., and Jastrzebska, M. (2011). “Immobilization techniques and biopolymer carriers,” Biotechnology and Food Science 75(1), 65-86.
Gülay, S., and Şanlı-Mohamed, G. (2012). “Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties,” International Journal of Biological Macromolecules 50(3), 545-551. DOI: 10.1016/j.ijbiomac.2012.01.017
Hoque, M. E., Shehryar, M., and Islam, K. M. N. (2013). “Processing and characterization of cockle shell calcium carbonate (CaCO3) bioceramic for potential application in bone tissue engineering,” Material Science and Engineering 2(4), 1-5. DOI: 10.4172/2169-0022.1000132
Muthuvel, I., Krishnakumar, B., and Swaminathan, M. (2012). “Novel Fe encapsulated montmorillonite K10 clay for photo-Fenton mineralization of Acid Yellow 17,” Indian Journal of Chemistry 51A, 800-806.
Sheldon, R. A. (2007). “Enzyme immobilization: The quest for optimum performance,” Advanced Synthesis & Catalysis 349, 1289-1307. DOI: 10.1002/adsc.200700082
Skels, P. (2011). “Quicklime (CaO) stabilization of fine-grained marine sediments in low temperature areas,” presented at 3rdInternational Conference Civil Engineering.
Talekar, S., and Chavare, S. (2012). “Optimization of immobilization alpha-amylase in alginate gel and its comparative biochemical studies with free alpha-amylase,” Recent Research in Science and Technology 4(2), 1-5.
Trindade, M. J., Dias, M. I., Coroado, J., and Rocha, F. (2009). “Mineralogical transformations of calcareous rich clays with firing: A comparative study between calcite and dolomite rich clays from Algarve, Portugal,” Applied Clay Science 42(3-4), 345-355. DOI: 10.1016/j.clay.2008.02.008
Tsai, W. T. (2013). “Microstructural characterization of calcite-based powder materials prepared by planetary ball milling,” Materials 6, 3361-3372. DOI: 10.3390/ma6083361
Witoon, T. (2011). “Characterization of calcium oxide derive from waste eggshell and its application as CO2 sorbent,” Ceramics International 37(8), 3291-3298. DOI: 10.1016/j.ceramint.2011.05.125
Zhou, Z.-d., Li, G.-y., and Li, Y.-j. (2010). “Immobilization of Saccharomyces cerevisiae alcohol dehydrogenase on hybrid alginate-chitosan beads,” International Journal of Biological Macromolecules 47(1), 21-26. DOI: 10.1016/j.ijbiomac.2010.04.001
Article submitted: August 4, 2014; Peer review completed: September 27, 2014; Revised version received and accepted: October 13, 2014; Published: October 20, 2014.