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MgO-Fe3O4 linked cellulase enzyme complex improves the hydrolysis of cellulose from Chlorella sp. CYB2
Accepted Manuscript Title: MgO-Fe3 O4 linked cellulase enzyme complex improves the hydrolysis of cellulose from Chlorella sp. CYB2 Authors: Rajendran Velmurugan, Aran Incharoensakdi PII: DOI: Reference:
S1369-703X(17)30057-8 http://dx.doi.org/doi:10.1016/j.bej.2017.02.012 BEJ 6662
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
2-12-2016 22-2-2017 26-2-2017
Please cite this article as: Rajendran Velmurugan, Aran Incharoensakdi, MgO-Fe3O4 linked cellulase enzyme complex improves the hydrolysis of cellulose from Chlorella sp.CYB2, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2017.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MgO-Fe3O4 linked cellulase enzyme complex improves the hydrolysis of cellulose from Chlorella sp. CYB2 RajendranVelmurugan, Aran Incharoensakdi* Cyanobacterial Biotechnology Laboratory Department of Biochemistry Faculty of Science Chulalongkorn University, Bangkok 10330, Thailand *Corresponding Author E-mail: [email protected] (A. Incharoensakdi); [email protected] (R. Velmurugan)
Graphical abstract Xylan O
O
OH
O HO
O
HO
O O
OH
O
HO OH
NaIO 4 Xylan aldehyde O
O
O
O
O
O
O O
O
O
O
O O
Coating on MgO-Fe3O4
MgO-Fe3O 4 and cellulase linked by xylan aldehyde
O
NH2-E (cellulase) -H2O
O
O O O
O
O
N
E
O CH
MgO-Fe3O 4 O O
O
O
CH
E
N E
O
N
H C
O
O HC
O
O
N E
Cellulose
Glucose
Graphical abstract Synthesis of xylan aldehyde for linking MgO-Fe3O4 and cellulase to hydrolyse cellulose into glucose.
1
Research Highlights Xylan aldehyde acts as a linking molecule for nanoparticle and enzymes MgO-Fe3O4 improved the recovery, hydrolysability and residual activity Linking of enzyme to nanoparticle affects the nature of α-helix and ß-sheet Maximum glucose yield of 91% was observed from Chlorella sp. CYB2
ABSTRACT In the present study, a novel catalyst was prepared by combining cellulase enzyme complex with metal oxides as a substrate activating and enzyme stabilizing component. The cellulose content obtained from Chlorella sp. CYB2 was treated with magnetic metal oxide linked enzyme complex through xylan aldehyde linking molecule. In order to improve the hydrolysis process, MgO was blended with Fe3O4 and the resulting complex had significant improvement in immobilization yield, activity recovery and hydrolysis of cellulose. Addition of xylan aldehyde as a linking molecule enhanced the enzyme binding onto metal nanoparticle. The results on hydrolysis showed the reduction in crystallinity of cellulose corresponding to the increase in enzymatic digestibility of cellulose. Under optimized conditions, the glucose yield obtained was 91% of theoretical maximum. The enhancement in 2
hydrolysis is correlated with the degradation of large molecule into well accessible substrate assisted by the action of metal oxides. Keywords: MgO, magnetic nanoparticle, xylan aldehyde, cellulase, hydrolysis
1. Introduction Biofuel production from non-edible biomass is an important issue that attracts considerable attention from researchers to meet the challenges posed by fossil fuels [1, 2]. The plant like photosynthetic microorganisms, especially microalgae, have gained much interest with regard to the production of biofuel from direct carbon fixation [3]. On the other hand, the algae could be cultivated to obtain the components such as starch, cellulose and lipids for biofuel production [2, 4]. The utilization of polysaccharide enriched microalgae for ethanol production can be economically feasible because of their simple growth, smaller particle size, amorphous nature and high polysaccharide with less phenolic content [5]. The microalga, Chlorella, has the ability to grow under both heterotrophic and phototrophic conditions and can accumulate about 40-70 % carbohydrate, which makes the algal biomass a potential feedstock for fermentable sugar production [6,7]. In most of the Chlorella, cellulose is reported as a major component, which is hydrolysed by cellulase to produce fermentable sugar [6,7]. Cellulase is a enzyme complex, each component of enzyme plays a vital role in hydrolysis. In general, enzymes have been used in industries for hydrolysis purposes but the enzyme requirement and inhibition makes the process unfeasible with regard to cellulosic ethanol production [8,9]. The cost of enzyme production and purification accounts for major production cost, which occupy a significant portion of sugar cost [8,9]. The immobilization methods such as cross-linking, binding to a support, and encapsulation or entrapment have 3
been performed to reuse the enzymes in the last few decades, in which various carriers made of organic or inorganic compounds have been studied [10]. Among the immobilization techniques, covalent binding is proved as an effective immobilization technique. Immobilization of enzyme onto charged magnetic metal oxides has more advantages over direct use of enzymes due to their recovery [10]. In case of cellulases, the magnetic metal oxide Fe3O4 has been frequently applied in various studies due to their easy recovery [10]. Abraham et al. [11] prepared magnetic nanoparticle for immobilization of cellulase and reported higher hydrolytic efficiency in immobilized enzyme compared to the free enzyme. On the other hand, metal oxides were used to suppress the inhibitory effect of phenolics, volatile acids and lignin [12]. For example, Ca2+ and Mg2+ were used to reduce or eliminate non-productive enzyme adsorption by phenolics and metal complex formation [13]. Azman et al. [12] reported that the presence of Mg2+ and Fe3+ ions in hydrolysis medium mitigated the inhibition of humic acid which is naturally produced during degradation of cellulosic biomass. To promote magnetic susceptibility and to provide large surface area for enzyme molecule, the aldehyde activated chitosan, starch, cellulose, polyethylene glycol and hyaluronic acid have been used [14]. It is also reported that the presence of polymer support between the metal oxide and enzyme provides freedom of movement which enhances the mobility of enzyme and reduces the hindrance [14]. Recently, Xu et al. [15] immobilized cellulase cocktail onto magnetic nanoparticles using glutaraldehyde cross linking and reported that the immobilized enzymes are effective for storage stability under a wide range of pH and temperature. On the other hand, metal oxides were used for direct conversion of polysaccharides into sugars at higher temperature [16]. Metal oxides in various forms have been used directly for hydrolysis purpose such as MgO in pretreatment of biomass [17], different metal oxides for fractionation of cyanobacterial biomass [18], Zn-Ca-Fe for cellulose hydrolysis [19] and carbon supported Ru for cellulose hydrolysis [16].The 4
development of an enzymatic hydrolysis process utilizing the chemical reaction at elevated temperature may facilitate a cascade system performing structural destabilization of polysaccharide and simultaneous enzymatic hydrolysis of carbohydrate fraction. Accordingly, this study was designed to prepare the metal oxide linked enzyme for improved performance, in which the mechanical insight into MgO on hydrolysis, xylan aldehyde on enzyme binding, binary nanoparticle (MgO-Fe3O4) on activity recovery and overall hydrolytic efficiency were analysed. The characteristics of metal oxide components were analysed by field emission scanning electron microscopy – energy dispersive X-ray spectroscopy (FESEM-EDS), whereas the secondary structures of enzymes were analysed by circular dichroism spectroscopy. Besides, the conjugation of enzymes onto the metal oxides, hydrolysis of Chlorella sp. CYB2 and activity recovery during hydrolysis were optimized. The novelties of this study are the demonstration of the use of MgO-Fe3O4 in increasing enzymatic hydrolysis, and more importantly the use of xylan aldehyde in linking cellulase onto binary metal nanoparticle (MgO-Fe3O4 ) with an improvement in hydrolysis even at a very high temperature. 2. Materials and Methods 2.1. Materials The metal oxides such as MgO (QRec®,New Zealand), ferric chloride (Himedia Laboratories Ltd) and ferrous chloride (Himedia Laboratories Ltd) were analytical grade. All the standards used for high performance liquid chromatography (HPLC) analysis were the products of Sigma Aldrich. The cellulase cocktail used in experiments is a mixture of commercial βglucosidase (Sisco Research Lab, India) and cellulase crude extract from Trichoderma reesei (MTCC 164) with the total enzyme unit ratio of 240 U β-glucosidase per FPU activity. The 5
purification was performed to prepare xylan-free cellulase by precipitation with (NH4)2SO4, molecular weight filtration (Sartorius, Vivacell 100, 30 kDa cut-off membrane) and then SDS-PAGE was performed (Fig. S2) [20]. 2.2.
Microorganism and cultivation conditions
The isolated Chlorella sp. CYB2 (Accession number: KP972095) was maintained in 250 mL Erlenmeyer flasks containing 100 mL BG-11 medium (pH 7.0). The cells were cultivated in 20 L reactor containing 15 L BG-11 medium (0.4%, w/v glucose supplementation) with a continuous illumination of 100 µmol photon/m2/s at 28 ± 1 °C for 20 days. Atmospheric CO2 was supplemented through 0.45 µm filter by air bubble method at the flow rate of 100 mL/min. After the cultivation, cells were harvested by centrifugation at 6000 × g for 10 min. Wet cells were immediately freeze dried before using for hydrolysis process [21]. The freeze dried biomass has the characteristic of forming slurry like precipitate with water. 2.3.
Nanoparticle linked enzyme preparation
The nanocatalyst linked enzyme was prepared by adding binary metal nanoparticle in enzyme solution, in which oxidized birchwood xylan (BWX) was used to make strong interaction between binary metal nanoparticle and cellulase enzyme complex. Fe3O4 particles were prepared by chemical co-precipitation of ferric chloride and ferrous chloride (1:2 ratio) in alkaline solution (pH 10) under constant stirring at 40 °C. The suspension was heated at 90 °C for 3 h and the obtained particles were separated by centrifugation at 5000 × g for 5 min and washed several times in water and finally with anhydrous ethanol. After oven drying at 70 °C, the MgO was added at different concentration (100 to 500 mg/g) to Fe3O4 and were heated at 90 °C for 3 h and the binary metal nanoparticles were coated with pre-prepared xylan aldehyde. The xylan aldehyde was prepared by dissolving 5 g of BWX in 100 mL of 250 mM sodium periodate. The solution was stirred for 10 h under dark condition at room 6
temperature. The precipitate of BWX aldehyde was purified by dialysis against ultrapure water after centrifugation at 8000 × g for 10 min. In coating process, BWX aldehyde was added into 100 mL distilled water, followed by dissolution with 1 g of urea and 1.5 g NaOH, and then stirred vigorously for 10 min. The xylan aldehyde solution was prepared in various concentrations to make 20–100 mg/g of MgO-Fe3O4 and the concentration of MgO-Fe3O4 was 2% (w/v). Finally, the solution was mixed with 8% (w/v) ammonium sulphate solution under constant stirring and the resulting precipitate was washed several times with distilled water and then dried at 60 °C in an oven for 5 h. The binding of binary metal nanoparticle with cellulase was carried out by immersing 1 g of xylan coated MgO-Fe3O4 in 50 mL solution of cellulase cocktail (mixture of endo-glucanase, cellobiohydrolase and βglucosidase in terms of protein at 50–250 mg/g). The solution was kept in a shaking incubator at 50 °C for 30 min and the precipitates obtained were washed with ultrapure water to remove unbounded enzyme and then the resulting products were used to determine the hydrolysability. The hydrolysis was carried out in 125 mL Erlenmeyer flask containing 50 mL of water, 3.33 g of biomass and 1 g of catalyst. The reaction was carried out at 60 °C with shaking at 200 rpm for 12 h, and then the solution was subjected to centrifugation at 8000 × g for 10 min to separate the enzyme linked nanoparticle from the hydrolysate. The upper liquid phase was used to determine the hydrolysed carbohydrate fraction, slurry like middle phase consisted of unhydrolysed biomass which was used for crystallinity index analysis, and the bottom thick solid phase was used to determine the immobilized enzyme and activity recoveries [22]. 2.4.
pH and thermal stability of immobilized enzyme during hydrolysis
The nanoparticle-linked cellulase prepared under optimised condition was added into a 125 mL Erlenmeyer flask containing 50 mL of water and 3.33 g biomass. The effects of pH (4.5, 7
5.0, 5.5, 6.0 and 6.5) and temperature (50, 55, 60, 65 and 70 °C) were analysed. After 12 h incubation with shaking at 200 rpm, the samples were centrifuged at 8000 × g for 10 min to separate the enzyme linked nanoparticle from the hydrolysate. The upper liquid phase was used to determine the glucose concentration and desorbed enzymes and the bottom thick solid phase was used to determine the immobilized enzyme and activity recoveries [22]. The percentage yield of glucose was calculated as the % conversion of available cellulose to glucose assuming the theoretical yield of 1.111 g/g cellulose [2]. 2.5.
Recycling of MgO-Fe3O4 linked enzyme catalyst and scale up studies
For recycling studies, 1 g of nanoparticle linked enzyme was used in 125 mL Erlenmeyer flask containing 50 mL of water and 3.33 g of biomass. Each hydrolysis cycle was run for 12 h at 60 °C and pH 5.5. To analyse the magnetic separation of immobilized enzyme, the immobilized enzymes were recovered using magnetic separation and the number of recycling was repeated up to seven cycles [22]. Briefly, 50 mL of hydrolysis medium containing immobilized enzyme was placed on magnetic separation stand (Lifesep 50SX) for 20 min to deposit the immobilized enzyme at the back wall. The supernatant was used for glucose yield whereas the deposited particles (immobilized enzyme) were washed twice with deionized water under magnetic separation. For scale up studies, the volume was increased to 1 L (1 L water, 66.6 g biomass and 20 g catalyst) and the rest were maintained under optimized conditions. 2.6.
Analytical methods
2.6.1. Physical characterization of catalyst and biomass The structure, size, shape and elemental composition of the MgO-Fe3O4 were characterized using field emission scanning electron microscope (FESEM) from CARL ZEISS, Germany and energy dispersive spectroscope (EDS) from Oxford instruments, United Kingdom. 8
Binding of enzyme onto nanoparticle was characterized by Fourier-transform infrared spectroscopy (FTIR) analyser (Perkin-Elmer FTIR spectrophotometer 2000 series) with the detector range at 4 cm-1 resolution and 25 scans per sample. The spectra were recorded between 4000 and 500 cm-1 [20]. The crystalline nature of the biomass was analysed using a Rigaku RINT-TTR3 X-ray diffractometer (Rigaku Co., Tokyo, Japan). The nickel-filtered CuKα radiation (k = 0.1542 nm) was applied at 50 kV and 30 mA. Samples were scanned over the range of 2h = 10–50 and the crystallinity index (CrI) was determined using Eqn. (1): [20]. CrI
I Crystalline I Amorphous I Crystalline
100 %
(1)
where, Icrystaline = intensity at 22°and Iamorphous= intensityat 18.8°. 2.6.2. Chemical characterization of hydrolysate The sugars such as glucose, galactose, mannose, xylose and arabinose were quantified using HPLC system (Shimadzu, Japan) equipped with refractive index detector (RID 10A, Shimadzu, Japan). The sugars were separated in Phenomenex, Rezex ROA-Organic acid column (150 × 7.8 mm) by using 5 mM H2SO4 as a mobile phase at a flow rate of 0.6 mL/min [23]. Polysaccharide contents in biomass were determined by acid hydrolysis method and the theoretical factors 1.111 and 1.136 were used for hexose and pentose sugars, respectively [2,24]. Total phenolics in biomass were determined by Folin-Ciocalteu method, using vanillin as a standard [25]. Protein content was estimated according to the method of Bradford using bovine serum albumin as standard [26]. The degree of oxidation of xylan was estimated using the method described by Zhao and Heindel [27]. Briefly, 0.1 g xylan aldehyde was suspended in 25 mL of 250 mM hydroxylamine hydrochloride solution, and then the pH was adjusted to 4.0 with 100 mM NaOH. The solution was stirred at room 9
temperature for 2 h, and then the suspension was titrated back to pH 4.0 with 100 mM NaOH. The degree of oxidation was calculated from the amount of NaOH consumed. 2.6.3. Enzyme characterization The crude enzyme extract, enzyme cocktail and biomass hydrolysate solution were used to determine filter paper (FPase), carboxymethylcellulase (CMCase), β-glucosidase and xylanase activities. One unit of activity (U) is defined as the amount (μmol) of product formed per minute under standard assay conditions [28]. The Fpase activity in immobilized enzyme was determined by adding 50 mg of metal nanoparticle linked enzyme and 50 mg of filter paper into 1.5 mL of 0.05 M sodium citrate buffer (pH 4.8) followed by incubation at 50 °C for 60 min. Dinitrosalicylic acid reagent was then added to the reaction mixture and the supernatant obtained after centrifugation (8,000 x g, 10 min) was used to determine the activity colorimetrically at 540 nm. The effect of nanoparticle on secondary structure of cellulase was analysed using circular dichroism (CD) spectroscopy. For every CD spectroscopic analysis, purified individual endo-glucanase, cellobiohydrolase and βglucosidase (0.1 mg protein/mL) was bounded with nanoparticle under optimized condition and was used for hydrolysis as mentioned in hydrolysis section [23,29]. The Far-UV CD spectra were recorded with spectropolarimeter (Jasco J-810, Japan) at 190–250 nm wavelength regions. The average values of three scans were used as a mean residue ellipticity [θ] (deg cm2 dmol−1) and the scans were analysed for secondary structure prediction using the K2D3 Algorithm [29]. 2.7.
Statistical analysis
All experiments were performed in triplicate and the average values are reported. The average and standard deviation values were calculated using the respective functions 10
(AVERAGE, STDEV) available in Microsoft Excel and the maximum difference among the three values was less than 5% of the mean. Statistical significances between the parameter levels were evaluated using Duncan's multiple range test at an α level of 0.05 (P<0.05). 3.
Results and Discussion
3.1.
Composition of raw biomass
The oven dried Chlorella sp. CYB2 biomass contained mass fraction of 38.32% total carbohydrates, in which the sugar components are glucose, galactose, mannose, arabinose and xylose (Table 1). Cellulose (33.6%) was the major component of total carbohydrates (Table 1). The lipids (17.4%), proteins (25.4%), phenolics (4.0%), ash (2.4%) and moisture (3.6%) were also present in biomass. The high content of carbohydrate fraction makes Chlorella sp. CYB2 biomass as a potential feedstock for fermentable sugar production. The composition of Chlorella sp. CYB2 observed in this study meets the feedstock criteria for biofuels production reported in previous studies [6,7]. 3.2. Cross linking of cellulase onto xylan aldehyde coated MgO-Fe3O4 In this study, the mixed oxides of Fe3O4 and MgO were developed as a support material for cellulase. As can be seen in Fig. 1a, the increase in MgO concentration from 100 to 400 mg/g increased the immobilization yield from 40 to 60.7%, beyond that it was saturated. This might be due to the disruption of ionic interaction between Fe3O4, MgO and enzyme caused by excessive addition of MgO. The hydrolysability was increased from 44.7 to 63.3% with an increase in MgO to Fe3O4 ratio, which indicates the significant influence of MgO on enzyme reactivity. According to Blanch [2], metal oxides develop cationic or anionic surface charge and alter the deprotonated carboxyl and phenolic site present in cellulosic biomass, which selectively disturb the cellulase inhibitors present in the biomass [2]. As shown in Fig. 1a, the 11
values for activity recovery after hydrolysis were varied from 76.7 to 96.3%, which indicate that the presence of MgO in nanoparticle improved the recovery of Fpase, CMCase and βglucosidase activities (Table S1). Overall, the immobilization yield and activity recovery influenced the hydrolysability of Chlorella sp. CYB2 biomass. The aldehyde groups of organic molecules have been utilized for cross-linking between the supporting material and enzyme molecules [15]. In this study, the BWX aldehyde has been utilized to improve the immobilization yield, which actually carries the aldehyde content of about 2.1 mM/g. As presented in Fig.1b, the increase in xylan aldehyde content increased the immobilization yield and hydrolysability until 60 mg/g, thereafter no significant improvement was observed. The results clearly indicate that the increase of xylan aldehyde content promotes more binding of enzyme onto nanoparticles via the increased linkage between aldehyde groups on surface of nanoparticles and amino groups of enzyme molecules. Similar results were obtained by Namdeo and Bajpai [30] when they used two different concentrations of oxidised cellulose for immobilization of α-amylase onto magnetic nanoparticles. The higher concentration of aldehyde was found to be a better option for the attachment of enzyme in immobilization. On the other hand, the activity recovery after hydrolysis was constantly increased with an increase in xylan aldehyde concentration and it reached the maximum activity recovery of 100% at 100 mg/g xylan aldehyde. The improvement of cellulase activity recovery in the presence of aldehyde group has been reported by Alahakoon et al. [31] with the immobilization yield of 52.4%. In the present study, the maximum immobilization yield of 88% and glucose yield of 76% were obtained at xylan aldehyde concentration of 60 mg/g, which indicates the potential of xylan aldehyde for use in immobilizing the enzymes.
12
The effects of enzyme concentration on immobilization yield, hydrolysability and activity recovery were investigated and the results are shown in Fig. 1c. Although, the increase in enzyme concentration slightly decreased the immobilization yield, it increased the total enzyme concentration bound to metal nanoparticles (Table S1). The maximum immobilization yield of 88.2% was observed at 150 mg/g of xylan aldehyde coated metal nanoparticles. The enzyme to nanoparticle ratio from 50 to 150 mg/g increased the hydrolysability from 56% to 90% and thereafter it reached the saturation. The saturation of enzyme binding to aldehyde surface was also reported in horseradish peroxidase [32] suggesting that the binding saturation is common irrespective of proteins or aldehyde source used. Besides, the activity recovery after hydrolysis was also found to increase with an increase in enzyme concentration from 50 to 100 mg/g and after which no significant difference in further increase of enzyme concentration (Fig. 1c). 3.3.
Physical characteristics of nanoparticle linked to enzyme
The morphology and composition of Fe3O4, MgO-Fe3O4 and enzyme linked MgO-Fe3O4 were analysed by FESEM-EDS and the results are shown in Figs. 2a – 2i. The spectrum results (Figs. 2a, 2d and 2g) show the presence of metal components with minor impurities. As presented in Figs 2b, 2e and 2h, the MgO-Fe3O4 particles were oval in shape and the average size was increased to ≈34 nm in diameter from ≈ 0.18 nm. After binding with the enzyme complex, the particles remained discrete and had a diameter of ≈34 nm. Similar results were reported by Jordan et al. [22] for immobilization of cellulase with magnetic nanoparticles. Figures 2c, 2f and 2i represent the weight percentage of individual components. As presented in Fig. 2i, the composition of metal oxides in enzyme linked MgOFe3O4 was similar to the composition of initial starting materials. The increase in carbon and oxygen contents in enzyme linked metal oxide (Fig. 2i) compared to that in metal oxide (Fig. 13
2f) indicates the presence of enzymes and xylan aldehyde in metal oxide nanoparticles. Although the carbon content was much lower than that of metal, the carbon content was equivalent with the content of protein and xylan aldehyde added. The binding of cellulase onto nanoparticle assisted by xylan aldehyde was confirmed by FTIR spectroscopy analysis. The FTIR spectra in Fig. 3 represent the spectra of Fe3O4, MgOFe3O4 -xylan aldehyde and cellulase-xylan aldehyde-MgO-Fe3O4. A stretch in the peak at 648 cm-1 indicates the Fe-O bond and the major peaks at 512, 586 and 671 cm-1 indicate the presence of stretching vibration of Mg-O, which demonstrated that MgO was successfully loaded with Fe3O4.The strong characteristic peaks at 1497 cm−1 and 1636 cm−1 were related to the stretching of C-O and C = O groups, respectively). By comparing the spectra of the xylan aldehyde coated MgO-Fe3O4 and Fe3O4 nanoparticles, it is observed that the bond around 3356 cm-1 is more pronounced and broader in xylan aldehyde coated than in the Fe3O4, indicating the presence of xylan aldehyde shell. The peak at 1544 cm-1 and the broadening of 1237 cm-1 peak are likely due to the formation of amide bond resulting from the linking of cellulase onto xylan aldehyde coated MgO-Fe3O4 [22]. A peak at 1041 cm−1 in enzyme linked MgO-Fe3O4 resembles the peak of cellulase molecule [11]. This characteristic shift in the frequency of cellulase-bound nanoparticles from the xylan aldehyde coated MgOFe3O4 is suggestive of covalent bonding of cellulase onto the nanoparticle [11]. Hence the results of FTIR confirmed successful mediation of xylan aldehyde on linking MgO-Fe3O4 with enzyme molecules. The secondary structural changes of individual cellulase components (endoglucanase, cellobiohydrolase and β–glucosidase) after linking MgO-Fe3O4 with enzyme were analysed using circular dichroism spectroscopy and the results are shown in Figs. 4a and 4b. The ellipticities were varied in all enzyme molecules, which indicate the alteration in molecular 14
shape of the cellulase (Fig. 4a). The free enzymes showed broad ellipticity whereas this was decreased with the enzymes bound to nanoparticles. The secondary structure of endoglucanase, cellobiohydrolase and β-glucosidase contains 27, 31 and 44% of α-helix and 22, 17, 14% of β-sheet, respectively (Fig. 4b). The results on secondary structure of nanoparticle linked enzyme revealed that the α-helix values were increased in cellobiohydrolase and β-glucosidase, while it was constant in endoglucanase. The β-sheet values were also increased in cellobiohydrolase and β-glucosidase, while it was decreased in endoglucanase. Mishra and Sardar [33] analysed the secondary structural changes of cellulase immobilized on silver and gold nanoparticles. The authors proved that the retaining of 85% α-helix structure is a crucial factor for retaining its activity. In the present study, the α-helix values were increased in cellobiohydrolase and β-glucosidase correlating well with the high activity recovery of CMCase and β-glucosidase at 95.2 and 98.6%, respectively, which were in agreement with previous studies [34,35]. 3.4.
Functional properties of nanoparticles and cellulase
In order to distinguish the role of metal oxides and xylan aldehyde in immobilization yield, hydrolysis and activity recovery after hydrolysis, each component was employed for the enzyme linked nanoparticle preparation and hydrolysis of microalgal biomass (Table 2). The immobilization yield of nanoparticle to the enzyme was less in Fe3O4 (59%) compared to MgO (71%), whereas the mixed metal oxides showed higher efficiency of 78%. Xylan aldehyde coated nanoparticles showed highest immobilization yield of 91.5%, which indicates the significance of xylan aldehyde coating in the linking of enzyme molecule to metal oxide nanoparticles. To determine the structural destabilisation of biomass, individual biomass obtained from different treatments (Fe3O4, MgO, MgO-Fe3O4, enzyme, and metal nanoparticle linked enzyme) were analysed using XRD (Fig. S1) and the values are 15
represented in Table 2. The crystallinity index of biomass in all treatments were reduced (22.1% - 28.3%) when compared to the raw biomass (30.1%, data not shown), while the highest reduction of crystallinty (22.1%) was observed in metal nanoparticle linked enzymatic hydrolysis. Hence, the reduced crystallinity facilitates the accessibility of enzymes to the substrate during the hydrolysis of cellulose. This contention is well supported by the glucose yield of 91.4% from the nanoparticle linked enzyme compared to the glucose yield of 77.2% from the free enzyme. Oligosaccharides were also detected after treatment of cellulose with Fe3O4, MgO and MgO-Fe3O4 giving the oligosaccharide yield of 1.2, 0.9 and 1.5 g/L, respectively (no oligosaccharide was detected in the control without nanoparticle). The results indicate that the metal oxides themselves have the hydrolysing ability to digest the cellulose, in which the oligosaccharides remained in different oligomers. Xie et al. [17] used MgO as a solid alkali for structural destabilization of cellulose and reported the significant reduction in degree of polymerization during hydrolysis. Similarly, Kobayashi et al. [16] observed the degradation of 1–4 linkage of cellulose when ruthenium was used as a catalyst. From the results, it can be confirmed that each individual component plays an important role in the hydrolysis of cellulose. The activity recoveries of three enzymes (Fpase, CMCase and β-glucosidase) after different treatments (Fe3O4, MgO, MgO-Fe3O4, enzyme, and metal nanoparticle linked enzyme) were analysed to determine the stability of the enzymes. As can be seen in Table 2, the activity recoveries after hydrolysis were higher in MgO-Fe3O4 than in free enzyme, which indicates the stability of enzyme was improved when linking onto metal nanoparticle. However, the activity recovery was highest in enzyme linked to xylan aldehyde coated MgO-Fe3O4, which indicates that the properties of a linking molecule can also contribute to the stability of the enzyme.
16
3.5.
Effect of pH and temperature on hydrolysis and stability of nanoparticle linked
enzyme For cellulases, the optimum pH 4.8 has been used in most studies for experimental purposes [28,31,36,37]. In the present study, the effects of initial pH on hydrolysis, activity recovery and desorption were investigated using the pH from 4.5 – 6.5 and the results obtained are shown in Fig. 5a. The hydrolysability was increased with an increase in pH and the maximum hydrolysability observed was 89% at pH 5.5, thereafter it was declined possibly due to the deactivation of enzyme at higher pH. The maximum activity recovery in terms of Fpase was 98% at pH 5.5, which is similarly observed for CMCase and β-glucosidase activities (Table S2). To analyse the amount of retaining protein content, desorption was analysed. The protein desorption was decreased with an increase in pH and reached the minimum desorption of 3% at pH 5.5, after that it was increased. The results confirmed that the increase in pH is likely to leach the protein from nanoparticle. Gokhale and Lee [36] immobilized cellulase and observed the swelling of cellulase which started at pH 7.0 and continued till pH 10.5. The authors also concluded that the biocatalytic potential of cellulase in these pH ranges was highly doubtful [36]. In the present study, desorption was below 7.3% in all tested pHs and the maximum activity recovery of 97% was observed at pH 5.5. Considering the protein and activity recoveries, pH 5.5 was selected as an optimum. The linking of the enzyme to a supporting material can increase the thermal stability by stabilizing the weak ionic forces and hydrogen bonds and thus increasing the range of operating temperatures. However, the denaturation of enzyme and reduced activity is also possible with higher temperatures. The changes in hydrolysability, activity recovery and protein desorption were analysed at different temperatures and the results are depicted in Fig. 5b. The hydrolysability was increased with an increase in temperature and the maximum 17
hydrolysability observed was 91% at 60 °C, thereafter it was reduced to 79% at 70 °C. The protein desorption was not significantly affected at all temperatures until 65 °C, thus confirming the possibilities of recovering protein even at high temperature. The activity recovery was slightly altered with an increase in temperature and the maximum activity recovery was observed at 60 °C. As the thermal stability increases, the cellulase can act at higher temperature resulting in an easy solubilization of cellulosic biomass. 3.6. Recycling of nanoparticle linked enzyme molecule The reuse of recovered nanoparticle linked enzyme for hydrolysis is a crucial factor for economic efficiency of the hydrolysis with enzymes, since enzymes are more expensive than chemical agents. Immobilized enzymes were separated using magnetic separation and were subjected to seven cycles of hydrolysis to analyse their efficiency after every cycle. As presented in Fig. 6, the activity recovery in immobilized enzyme was slightly decreased after each cycle of hydrolysis. After seven cycles, about 84.5% activity was retained compared to about 97% at the start of the experiments. The decrease in activity might be due to both desorption and denaturation of enzymes. Similar reusability studies on immobilized cellulase have been reported. Gokhale et al [37] immobilized cellulase directly onto magnetic nanoparticles; however, the enzyme retained about 55% of original activity after four cycles of reuse. Alahakoon et al. [31] compared the use of amine and aldehyde functionalized Fe3O4 magnetic nanoparticles for cellulase immobilization and reported that aldehyde functionalized nanoparticle retained higher activity (76.5–87%) than amine functionalized nanopartilce (70.1–76.4%) after six cycles. In comparison, the present study achieved higher activity recovery than the research studies reported earlier. The improved activity recovery is mainly attributed to the flexible interaction between metal nanoparticle and enzyme molecule assisted by xylan aldehyde. It is noted that both the catalyst recovery and the glucose yield were relatively unaffected even after seven cycles of hydrolysis. 18
3.7.
Scale up studies on hydrolysis
The scale up studies were carried out by increasing the reaction volume to 1 L under optimized condition. The scale up studies up to 1 L showed almost 90% of glucose yield which confirms the application of nanoparticle linked enzymatic catalyst at a large scale. Hydrolysis of 100 g of algal biomass resulted in 22.5 g of glucose whereas the unhydrolysed carbohydrate, lipid and protein contents were recovered as a solid biomass. Overall, the glucose production of 0.336 g/g of original biomass was observed after saccharification and 0.034 g/g of unhydrolysed cellulose was retained in solid content. The quantity of immobilized enzyme added was 30 g per 100 g of biomass; however, the optimization of catalyst loading can reduce the catalyst requirement in commercialization. In general, the reaction of solid biocatalyst with solid substrate is low due to the poor accessibility of the enzyme to the substrate. In most of the previous studies the plant biomass was pretreated before hydrolysis using immobilized enzymes. The pretreatment renders the substrate present in the biomass more readily accessible to the enzyme. Kumakura [38] immobilized cellulase onto aluminium oxide beads and observed 53% hydrolysis of cellulose component of chaff, pretreated by electron beam irradiation. Similarly, the immobilization of cellulase onto cyclodextrin conjugated magnetic particle improved hydrolytic efficiency on rice straw after pretreatment by grinding into powder form [39]. Abraham et al. [11] immobilized cellulase for the hydrolysis of hump hurd biomass pretreated with NaOH and reported maximum hydrolysis of 93% in immobilized enzyme which is comparatively higher than free enzyme (89%). However, in this study most of the algal cellulose were converted to glucose, which indicates the potential of the developed immobilization method in algal biomass utilization. Unlike plant biomass, the algal biomass becomes more susceptible for hydrolysis, which is partly due to the reduced particle size and the amorphous nature of algal biomass compared 19
to plant biomass. However, a separate pretreatment process and a suitable immobilization method should be evaluated when using lignocellulosic plant biomass. The efficient recovery of immobilized enzyme and an improvement of hydrolysis make the process attractive with high potential for industrial utilization of algal biomass for fermentable sugar production.
4. Conclusion The development of a viable bioconversion process for fermentable sugar production is the major challenge in cellulosic ethanol production process. This study demonstrated that MgO improved the hydrolysability of biomass, whereas the Fe3O4 facilitated the separation of catalyst. Addition of xylan aldehyde improved the enzyme binding to the metal nanoparticle, without reducing the hydrolytic performance. Furthermore, the scale up study provides more insight into its commercialization potential. Acknowledgements R.V. is thankful to the Graduate School and Faculty of Science, Chulalongkorn University (CU), for post-doctoral fellowship from Rachadaphiseksomphot Endowment Fund. A.I. acknowledges the research grant from CU on the Frontier Research Energy Cluster (CU-59048-EN). Appendices Immobilization yield was obtained from immobilization experiments and was calculated from the following equation (Eq. A.1) Im mobilizati on yield
( I P LP ) 100 IP
(A.1)
where, LP is the concentration of protein observed in liquid (mg/50 mL), IP is the concentration of protein added (mg/50 mL).
20
Hydrolysability was determined after hydrolysis experiment and was calculated from the following equation (Eq. A.2) Glu cos e yield
Qglc (Qcellulose 1.111)
100
(A.2)
where, Qglc is the concentration of glucose observed in hydrolysate (g/50 mL), Qcellulose is the concentration of cellulose (quantity of biomass × 0.336) added (g/50mL), 1.111 is the theoretical factor. Activity recovery was determined after every hydrolysis experiment and was calculated from the following equation (Eq. A.3) Activity re cov ery
Factivity I ativity
100
(A.3)
where, Factivity is the total activity obtained after hydrolysis (U/ mL), Iactivity is the initial activity in immobilized enzyme (U/ mL). Desorption of protein after hydrolysis was calculated from the following equation (Eq. A.4)
Desorption
I protein Fprotein I protein
100
(A.4)
where, Iprotein is the concentration of nanoparticle linked protein added (mg/50 mL), Fprotein is the concentration of protein recovered along with nanoparticle (mg/50 mL). References [1] D.O. Hall, Biomass for energy – a worldwide view, In: E. Campos-Lopez (Ed), Renewable resources a systematic approach, Academic Press, USA, 1980, pp. 287–305. [2] H.W. Blanch, Bioprocessing for biofuels, Curr. Opin. Biotech. 23 (2012) 390–395. [3] P. Savakis, K.J. Hellingwerf, Engineering cyanobacteria for direct biofuel production from CO2, Curr. Opin. Biotechnol. 33 (2015) 8–14. [4] X. Zeng, M.K. Danquah, X.D. Chen, Y. Lu, Microalgae bioengineering: From CO2 fixation to biofuel production, Renew. Sust. Energ. 15 (2011) 3252–3260. 21
[5] M.S. Miranda, S. Sato, J. Mancini-Filho, Antioxidant activity of the microalga chlorella vulgaris cultured on special conditions, Boll. Chim. Farm. 140 (2001) 165-168. [6] N. Zhou, Y. Zhang, X. Gong, Q. Wang, Y. Ma, Ionic liquids-based hydrolysis of Chlorella biomass for fermentable sugars, Bioresour. Technol. 118 (2012) 512–517. [7] L. Brennan, P. Owende, Biofuels from microalgae - A review of technologies for production, processing, and extractions of biofuels and co-products, Renew. Sust. Energ. Rev. 14 (2010) 557–577. [8] M. Tu, R.P. Chandra, J.N. Saddler, Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates, Biotechnol. Prog. 23 (2007) 398–406. [9] M. Galbe, G. Zacchi, A review of the production of ethanol from softwood, Appl. Microbiol. Biotechnol. 59 (2002) 618–628. [10] M. Misson, H. Zhang, B. Jin. Nanobiocatalyst advancements and bioprocessing applications, J. R. Soc. Interface 12 (2015) 20140891.
[11] R.E Abraham, M.L. Verma, C.J. Barrow M. Puri. Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass. Biotech. Biofuel. 7 (2014) 90.
[12] S. Azman, A.F. Khadem, G. Zeeman, J.B. van Lier, C.M. Plugge, Mitigation of humic acid inhibition in anaerobic digestion of cellulose by addition of various salts, Bioeng. 2 (2015) 54-65. [13] H. Liu, J.Y. Zhu, Eliminating inhibition of enzymatic hydrolysis by lignosulfonate in unwashed sulfite-pretreated aspen using metal salts, Bioresour. Technol. 101 (2010) 9120– 9127.
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[14] H. Vaghari, H. Jafarizadeh-Malmiri, M. Mohammadlou, A. Berenjian, N. Anarjan, N. Jafari, S. Nasiri, Application of magnetic nanoparticles in smart enzyme immobilization, Biotechnol. Lett. 38 (2016) 223–233. [15] J.S. Xu, Z. Huo, Y. Yuan, H. Zhang, Y. Xu, C. Guo, Liang, X. Zhuang, Characterization of direct cellulase immobilization with super paramagnetic nanoparticles, Biocatal. Biotransfor. 29 (2011) 71–76. [16] H. Kobayashi, T. Komanoya, K. Hara, A. Fukuoka, Water-tolerant mesoporous-carbonsupported ruthenium catalysts for the hydrolysis of cellulose to glucose, Chem. Sus. Chem. 3 (2010) 440–443. [17] T. Xie, L. Lin, C. Panga, J. Zhuanga, J. Shia, Q. Yang, Efficient enzymatic hydrolysis of the bagasse pulp prepared with active oxygen and MgO-based solid alkali, Carbohydr. Polym. 94 (2013) 807–813. [18] R. Velmurugan, A. Incharoensakdi, Potential of metal oxides in fractionation of Synechocystis sp. PCC 6803 biomass for biofuel production, Algal Res. 19 (2016) 96–103. [19] F. Zhang, X. Deng, Z. Fang, H.Y. Zeng, X.F. Tian, J.A. Kozinski, Hydrolysis of crystalline cellulose over Zn-Ca-Fe oxide catalyst, Petrochem. Technol. 40 (2011) 43–48. [20] R. Velmurugan, A. Incharoensakdi, Proper ultrasound treatment increases ethanol production from simultaneous saccharification and fermentation of sugarcane bagasse, RSC Adv. 6 (2016) 91409-91419. [21] T. Monshupanee, A. Incharoensakdi, Enhanced accumulation of glycogen, lipids and polyhydroxybutyrate under optimal nutrients and light intensities in the cyanobacterium Synechocystis sp. PCC 6803, J. Appl. Microbiol. 116 (2013) 830-838. [22] J. Jordan, C.S.S.R. Kumar, C. Theegala, Preparation and characterization of cellulasebound magnetite nanoparticles, J. Mol. Catal. B: Enzym. 68 (2011) 139-146.
23
[23] R. Velmurugan, K. Muthukumar, Sono-assisted enzymatic saccharification of sugarcane bagasse for bioethanol production, Biochem. Eng. J. 63 (2012) 1-9. [24] R. Ruiz, T. Ehrman, Determination of carbohydrates in biomass by high performance liquid chromatography, NREL protocol/LAP-002, Golden, CO, USA, 1996. [25] V.L. Singleton, R. Orthofer, R.M. Lamuela-Raventos, Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent, Methods Enzymol. 299 (1999) 152-178. [26] M. Bradford, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 27 (1976) 248-254. [27] H. Zhao, N.D. Heindel, Determination of degree of substitution of formyl groups in polyaldehyde dextran by the hydroxylamine hydrochloride method, Pharmaceut. Res. 8 (1991) 400-402. [28] T. K. Ghose. Measurement of cellulase activities, Pure. Appl. Chem. 59 (1987) 257-268. [29] C. Louis-Jeune, M.A. Andrade-Navarro, C. Perez-Iratxeta, Prediction of protein secondary structure from circular dichroism using theoretically derived spectra, Proteins: Struct, Funct. Bioinformat. 80 (2012) 374-381. [30] M. Namdeo, S.K. Bajpai, Immobilization of α-amylase onto cellulose-coated magnetite (CCM) nanoparticles and preliminary starch degradation study, J. Mol. Catal. B. Enzym. 59 (2009) 134–139. [31] T. Alahakoon, J.W. Koh, X.W.C. Chong, W.T.L. Lim, Immobilization of cellulases on amine and aldehyde functionalized Fe2O3 magnetic nanoparticles, Prep. Biochem. Biotechnol. 42 (2012) 234–248. [32] N. Isobe, D.S. Lee, Y.J. Kwon, S. Kimura, S. Kuga, M. Wada, U.J. Kim, Immobilization of protein on cellulose hydrogel, Cellulose. 18 (2011) 1251.
24
[33] A. Mishra, M. Sardar, Cellulase assisted synthesis of nano-silver and gold: Application as immobilization matrix for biocatalysis, Int. J. Biol. Macromol. 77 (2015) 105–113. [34] Z. Wu, B. Zhang, B. Yan. Regulation of enzyme activity through interactions with nanoparticles, Int. J. Mol. Sci. 10 (2009) 4198–4209. [35] S. Zolghadri, A.A. Saboury, E. Amin, A.A. Moosavi-Movahedi, A spectroscopic study on the interaction between ferric oxide nanoparticles and human haemoglobin, J. Iran Chem. Soc. 7 (2010)145–153. [36] A.A. Gokhale, I. Lee, Cellulase immobilized nanostructured supports for efficient saccharification of cellulosic substrates, Top. Catal. 16 (2012) 1231–1246. [37] A.A. Gokhale, J. Lu, I. Lee, Immobilization of cellulase on magneto responsive graphane nano-supports, J. Mol. Catal. B: Enzym. 90 (2013) 76–86. [38] M. Kumakura, Preparation of immobilized cellulase beads and their application to hydrolysis of cellulosic materials, Process Biochem. 32 (1997) 555-559. [39] P.J. Huang, K.L. Chang, J.F. Hsieh, S.T. Chen, Catalysis of rice straw hydrolysis by the combination of immobilized cellulase from Aspergillus niger on β-cyclodextrin-Fe3O4. nanoparticle and ionic liquid, BioMed Res. Int. (2015) 409103,
doi.org/10.1155/2015/409103.
Figure Captions Fig. 1 Factors affecting immobilization yield, activity recovery and glucose yield (a) MgO to Fe3O4 ratio, (b) xylan aldehyde concentration, (c) protein concentration. Symbols: ■immobilization yield ■-activity recovery, □- glucose yield. Different letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05. 25
Fig. 2 FESEM-EDS analysis of Fe3O4 (a-spectrum, b-microscopic image, c-weight percentage), xylan aldehyde coated MgO-Fe3O4 (d-spectrum, e-microscopic image, f-weight percentage), xylan aldehyde coated MgO-Fe3O4 linked to enzyme (g-spectrum, h-microscopic image, i-weight percentage). Fig. 3 Fourier transform infrared spectra of (a) Fe3O4 (b) xylan aldehyde coated MgO-Fe3O4, (c) enzyme linked xylan aldehyde coated MgO-Fe3O4. Fig. 4 (a) Circular dichroism spectrum (symbols: (1) EG control (2) EG- MgO-Fe3O4 (3) CBH
control (4) CBH- MgO-Fe3O4 (5) BGL-control (6) BGL- MgO-Fe3O4) and (b) Percentage αhelix and β-sheet values (symbols: ■-α helix and □-β sheet) of endoglucanase I (EG: 46 kDa), cellobiohydrolase I (CBH: 56.2 kDa) and β-glucosidase (BGL: 75 kDa) for control (without MgO-Fe3O4) and MgO-Fe3O4 linked enzyme. Different letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05. Fig. 5 Effect of temperature on glucose yield, residual activity and desorption of protein. Symbols: ■-glucose yield, ■-activity recovery, □-desorption. Different letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05. Fig. 6 Recycling of metal nanoparticle linked enzyme and glucose yield after each cycle of hydrolysis. Symbols: ■-catalyst recovery, ■- glucose yield, □- activity recovery. Control represents the analysis at the start of the experiments. Different letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05.
26
Immobilization yield (%); activity recovery (%); glucose yield (%)
(a)
120 100
d
e
80
b
60 40
d
e
d
a
b
a
a
a
c
20 0 Control
100
200 300 400 MgO to Fe3O4 ratio (mg/g)
(b) 120
Immobilization yield (%); activity recovery (%); glucose yield (%)
c
d
a
a,b
b
c
100
b
80
c
c
a a
a
60 40 20
0 20
40 60 80 Xylan aldehyde concentration (mg/g)
(c) 120 Immobilization yield (%); activity recovery (%); glucose yield (%)
a
a
b
a
b
a b,c
c
c
500
100
a
a
c
80
a
a
a
a,b
b
100
a
a
a
b
b c
c
60 40 20 0 50
100 150 200 Protein concentration (mg/g)
250
Fig. 1
27
(c) 80 Weight percentage (%)
(b)
(a)
Weight percentage (%)
80 60 40
20
60 40
20
0 C
O
30 µm
(d)
S Fe Components Electron Image 1
(e)
Weight percentage (%)
(f)
Weight percentage (%)
80 60 40
20 0 C
O
30 µm
S Fe Components Electron Image 1
O
S Fe Components
Mg
C
O
S Fe Components
Mg
C
O
S Fe Components
Mg
80
60 40
20 0
(i) Weight percentage (%)
80
Weight percentage (%)
80 60 40
20 0 C
KeV
C
Mg
(h)
(g)
0
Mg
30 µm
O
S Fe Components Electron Image 1
60 40
20
Mg
0
Fig. 2
28
Transmittance Transmittance (not (not for scale)
(a) (a) (b) (b) (c)
1636 1497 1544 1237 1041
3356
671 512 586
4000 4000
3500 3500
3000 3000
2500 2000 1500 2500 2000 1500 Wave number (cm-1) Wave number (cm-1)
1000 1000
500 500
Fig. 3
29
(a)
10
(6) (5)
8
(4) (3)
[θ] (103 degcm-2 dmol -1)
6 4 2
(2) (1)
0 -2 -4 -6 -8 190
200
210 220 Wavelength (nm)
230
240
(b) 50
a
a
45
α-helix and β-sheet values (%)
40 35 30 25 20
b
c d
d a
b c
15
c
e
d
10 5 0 EG EG CBH CBH BGL BGL (Control) (MgO-Fe (MgO-3O4) (Control) (MgO-Fe (MgO(Control) (MgOO ) (MgO-Fe 3 4 3O4) Fe3O4) Fe3O4) Fe3O4)
Enzymes
Enzymes
Fig. 4
30
Glucose yield (%); activity recovery (%); desorption (%)
(a) 120 a
100
b
c 80
a
a
a
b b
c
c
60 40 20
a
a
a
a
a
0 4.5
5.5
6.0
6.5
a
a
pH 120
Glucose yield (%); activity recovery (%); desorption (%)
(b)
5.0
a
a
100
a a
b
c
b
c
80 60
40 20
b
b
b
a
b
0 50
55
60 65 Temperature (°C)
70
Fig. 5
31
Immobilized enzyme recovery (%); glucose yield (%); activity recovery (%)
120
100
a aa
1
a
aa
2
a a a
a a a
a a a
3 4 5 Number of cycles
a
a
6
a
a a
b
80
60
40
20
0
7
Fig. 6
32
List of Tables Table 1 Composition of Chlorella sp. CYB2 Components
Concentration (%, w/w)
Cellulose
33.6±0.78
Galactan
1.8±0.49
Manosan
0.9±0.38
Arabinan
1.1±0.32
Xylan
1.0±0.41
Lipids
17.3±0.70
Protein
25.3±0.67
Phenolics
4.0±0.89
Ash
2.4±0.55
Moisture
3.6±0.78
Other
9±1.32
33
Table 2 Comparison of individual and immobilized catalyst on immobilization yield, activity recovery, and hydrolysis of Chlorella sp. CYB2 cellulose content. Activity recovery b Immobiliz Compon ation yield a ent
FPase
CMCa se
βglucosi dase
(g/g, %)
Crystall Hydrolytic efficiencyc inty index glucos cellobi oligo- glucose sacchar of e ose yield ide biomas (g/L) (g/L) (%) (g/L) s (%)
Fe3O4
59.4±2.1d
76.0± 2.5c
45.8± 4.4d
74.7±1. 5d
28.3±1. 4a
0.1±0. 0c
0.7±0. 1b
1.2±0. 1a
1.0±0. 1c
MgO
71.5±4.4c
91.1± 0.6b
99.4± 4.3a
92.8±1. 4b
26.4±2. 2b
0.1±0. 1c
0.5±0. 2b
0.9±0. 3a,b
1.0±0. 3c
MgOFe3O4
77.6±3.9b
78.1± 1.8c
62.8± 2.6b
85.3±1. 7c
24.8±1. 6c
0.2±0. 0c
0.9±0. 2b
1.5±0. 3a
1.4±0. 2c
Enzy me
na
76.2± 4.6c
59.7± 3.2c
75.2±2. 2d
27.6±0. 7a,b
14.5± 0.4b
1.3±0. 4a
0.3±0. 4b
77.2± 2.1b
Enzy meXylan aldehy deMgOFe3O4
91.5±3.5a
97.6± 1.9a
95.2± 1.1a
98.6±1. 2a
22.1±2. 1d
17.1± 0.3a
0.8±0. 3b
0.2±0. 1b
91.4± 1.5a
na-not applicable. a immobilization yield was analysed by adding the protein concentration of 150 mg/g metal oxide; b activity recovery was determined after hydrolysis; c hydrolytic efficiency was carried out with individual component as mentioned in first row. The superscript letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05.
34
S1369-703X(17)30057-8 http://dx.doi.org/doi:10.1016/j.bej.2017.02.012 BEJ 6662
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
2-12-2016 22-2-2017 26-2-2017
Please cite this article as: Rajendran Velmurugan, Aran Incharoensakdi, MgO-Fe3O4 linked cellulase enzyme complex improves the hydrolysis of cellulose from Chlorella sp.CYB2, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2017.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MgO-Fe3O4 linked cellulase enzyme complex improves the hydrolysis of cellulose from Chlorella sp. CYB2 RajendranVelmurugan, Aran Incharoensakdi* Cyanobacterial Biotechnology Laboratory Department of Biochemistry Faculty of Science Chulalongkorn University, Bangkok 10330, Thailand *Corresponding Author E-mail: [email protected] (A. Incharoensakdi); [email protected] (R. Velmurugan)
Graphical abstract Xylan O
O
OH
O HO
O
HO
O O
OH
O
HO OH
NaIO 4 Xylan aldehyde O
O
O
O
O
O
O O
O
O
O
O O
Coating on MgO-Fe3O4
MgO-Fe3O 4 and cellulase linked by xylan aldehyde
O
NH2-E (cellulase) -H2O
O
O O O
O
O
N
E
O CH
MgO-Fe3O 4 O O
O
O
CH
E
N E
O
N
H C
O
O HC
O
O
N E
Cellulose
Glucose
Graphical abstract Synthesis of xylan aldehyde for linking MgO-Fe3O4 and cellulase to hydrolyse cellulose into glucose.
1
Research Highlights Xylan aldehyde acts as a linking molecule for nanoparticle and enzymes MgO-Fe3O4 improved the recovery, hydrolysability and residual activity Linking of enzyme to nanoparticle affects the nature of α-helix and ß-sheet Maximum glucose yield of 91% was observed from Chlorella sp. CYB2
ABSTRACT In the present study, a novel catalyst was prepared by combining cellulase enzyme complex with metal oxides as a substrate activating and enzyme stabilizing component. The cellulose content obtained from Chlorella sp. CYB2 was treated with magnetic metal oxide linked enzyme complex through xylan aldehyde linking molecule. In order to improve the hydrolysis process, MgO was blended with Fe3O4 and the resulting complex had significant improvement in immobilization yield, activity recovery and hydrolysis of cellulose. Addition of xylan aldehyde as a linking molecule enhanced the enzyme binding onto metal nanoparticle. The results on hydrolysis showed the reduction in crystallinity of cellulose corresponding to the increase in enzymatic digestibility of cellulose. Under optimized conditions, the glucose yield obtained was 91% of theoretical maximum. The enhancement in 2
hydrolysis is correlated with the degradation of large molecule into well accessible substrate assisted by the action of metal oxides. Keywords: MgO, magnetic nanoparticle, xylan aldehyde, cellulase, hydrolysis
1. Introduction Biofuel production from non-edible biomass is an important issue that attracts considerable attention from researchers to meet the challenges posed by fossil fuels [1, 2]. The plant like photosynthetic microorganisms, especially microalgae, have gained much interest with regard to the production of biofuel from direct carbon fixation [3]. On the other hand, the algae could be cultivated to obtain the components such as starch, cellulose and lipids for biofuel production [2, 4]. The utilization of polysaccharide enriched microalgae for ethanol production can be economically feasible because of their simple growth, smaller particle size, amorphous nature and high polysaccharide with less phenolic content [5]. The microalga, Chlorella, has the ability to grow under both heterotrophic and phototrophic conditions and can accumulate about 40-70 % carbohydrate, which makes the algal biomass a potential feedstock for fermentable sugar production [6,7]. In most of the Chlorella, cellulose is reported as a major component, which is hydrolysed by cellulase to produce fermentable sugar [6,7]. Cellulase is a enzyme complex, each component of enzyme plays a vital role in hydrolysis. In general, enzymes have been used in industries for hydrolysis purposes but the enzyme requirement and inhibition makes the process unfeasible with regard to cellulosic ethanol production [8,9]. The cost of enzyme production and purification accounts for major production cost, which occupy a significant portion of sugar cost [8,9]. The immobilization methods such as cross-linking, binding to a support, and encapsulation or entrapment have 3
been performed to reuse the enzymes in the last few decades, in which various carriers made of organic or inorganic compounds have been studied [10]. Among the immobilization techniques, covalent binding is proved as an effective immobilization technique. Immobilization of enzyme onto charged magnetic metal oxides has more advantages over direct use of enzymes due to their recovery [10]. In case of cellulases, the magnetic metal oxide Fe3O4 has been frequently applied in various studies due to their easy recovery [10]. Abraham et al. [11] prepared magnetic nanoparticle for immobilization of cellulase and reported higher hydrolytic efficiency in immobilized enzyme compared to the free enzyme. On the other hand, metal oxides were used to suppress the inhibitory effect of phenolics, volatile acids and lignin [12]. For example, Ca2+ and Mg2+ were used to reduce or eliminate non-productive enzyme adsorption by phenolics and metal complex formation [13]. Azman et al. [12] reported that the presence of Mg2+ and Fe3+ ions in hydrolysis medium mitigated the inhibition of humic acid which is naturally produced during degradation of cellulosic biomass. To promote magnetic susceptibility and to provide large surface area for enzyme molecule, the aldehyde activated chitosan, starch, cellulose, polyethylene glycol and hyaluronic acid have been used [14]. It is also reported that the presence of polymer support between the metal oxide and enzyme provides freedom of movement which enhances the mobility of enzyme and reduces the hindrance [14]. Recently, Xu et al. [15] immobilized cellulase cocktail onto magnetic nanoparticles using glutaraldehyde cross linking and reported that the immobilized enzymes are effective for storage stability under a wide range of pH and temperature. On the other hand, metal oxides were used for direct conversion of polysaccharides into sugars at higher temperature [16]. Metal oxides in various forms have been used directly for hydrolysis purpose such as MgO in pretreatment of biomass [17], different metal oxides for fractionation of cyanobacterial biomass [18], Zn-Ca-Fe for cellulose hydrolysis [19] and carbon supported Ru for cellulose hydrolysis [16].The 4
development of an enzymatic hydrolysis process utilizing the chemical reaction at elevated temperature may facilitate a cascade system performing structural destabilization of polysaccharide and simultaneous enzymatic hydrolysis of carbohydrate fraction. Accordingly, this study was designed to prepare the metal oxide linked enzyme for improved performance, in which the mechanical insight into MgO on hydrolysis, xylan aldehyde on enzyme binding, binary nanoparticle (MgO-Fe3O4) on activity recovery and overall hydrolytic efficiency were analysed. The characteristics of metal oxide components were analysed by field emission scanning electron microscopy – energy dispersive X-ray spectroscopy (FESEM-EDS), whereas the secondary structures of enzymes were analysed by circular dichroism spectroscopy. Besides, the conjugation of enzymes onto the metal oxides, hydrolysis of Chlorella sp. CYB2 and activity recovery during hydrolysis were optimized. The novelties of this study are the demonstration of the use of MgO-Fe3O4 in increasing enzymatic hydrolysis, and more importantly the use of xylan aldehyde in linking cellulase onto binary metal nanoparticle (MgO-Fe3O4 ) with an improvement in hydrolysis even at a very high temperature. 2. Materials and Methods 2.1. Materials The metal oxides such as MgO (QRec®,New Zealand), ferric chloride (Himedia Laboratories Ltd) and ferrous chloride (Himedia Laboratories Ltd) were analytical grade. All the standards used for high performance liquid chromatography (HPLC) analysis were the products of Sigma Aldrich. The cellulase cocktail used in experiments is a mixture of commercial βglucosidase (Sisco Research Lab, India) and cellulase crude extract from Trichoderma reesei (MTCC 164) with the total enzyme unit ratio of 240 U β-glucosidase per FPU activity. The 5
purification was performed to prepare xylan-free cellulase by precipitation with (NH4)2SO4, molecular weight filtration (Sartorius, Vivacell 100, 30 kDa cut-off membrane) and then SDS-PAGE was performed (Fig. S2) [20]. 2.2.
Microorganism and cultivation conditions
The isolated Chlorella sp. CYB2 (Accession number: KP972095) was maintained in 250 mL Erlenmeyer flasks containing 100 mL BG-11 medium (pH 7.0). The cells were cultivated in 20 L reactor containing 15 L BG-11 medium (0.4%, w/v glucose supplementation) with a continuous illumination of 100 µmol photon/m2/s at 28 ± 1 °C for 20 days. Atmospheric CO2 was supplemented through 0.45 µm filter by air bubble method at the flow rate of 100 mL/min. After the cultivation, cells were harvested by centrifugation at 6000 × g for 10 min. Wet cells were immediately freeze dried before using for hydrolysis process [21]. The freeze dried biomass has the characteristic of forming slurry like precipitate with water. 2.3.
Nanoparticle linked enzyme preparation
The nanocatalyst linked enzyme was prepared by adding binary metal nanoparticle in enzyme solution, in which oxidized birchwood xylan (BWX) was used to make strong interaction between binary metal nanoparticle and cellulase enzyme complex. Fe3O4 particles were prepared by chemical co-precipitation of ferric chloride and ferrous chloride (1:2 ratio) in alkaline solution (pH 10) under constant stirring at 40 °C. The suspension was heated at 90 °C for 3 h and the obtained particles were separated by centrifugation at 5000 × g for 5 min and washed several times in water and finally with anhydrous ethanol. After oven drying at 70 °C, the MgO was added at different concentration (100 to 500 mg/g) to Fe3O4 and were heated at 90 °C for 3 h and the binary metal nanoparticles were coated with pre-prepared xylan aldehyde. The xylan aldehyde was prepared by dissolving 5 g of BWX in 100 mL of 250 mM sodium periodate. The solution was stirred for 10 h under dark condition at room 6
temperature. The precipitate of BWX aldehyde was purified by dialysis against ultrapure water after centrifugation at 8000 × g for 10 min. In coating process, BWX aldehyde was added into 100 mL distilled water, followed by dissolution with 1 g of urea and 1.5 g NaOH, and then stirred vigorously for 10 min. The xylan aldehyde solution was prepared in various concentrations to make 20–100 mg/g of MgO-Fe3O4 and the concentration of MgO-Fe3O4 was 2% (w/v). Finally, the solution was mixed with 8% (w/v) ammonium sulphate solution under constant stirring and the resulting precipitate was washed several times with distilled water and then dried at 60 °C in an oven for 5 h. The binding of binary metal nanoparticle with cellulase was carried out by immersing 1 g of xylan coated MgO-Fe3O4 in 50 mL solution of cellulase cocktail (mixture of endo-glucanase, cellobiohydrolase and βglucosidase in terms of protein at 50–250 mg/g). The solution was kept in a shaking incubator at 50 °C for 30 min and the precipitates obtained were washed with ultrapure water to remove unbounded enzyme and then the resulting products were used to determine the hydrolysability. The hydrolysis was carried out in 125 mL Erlenmeyer flask containing 50 mL of water, 3.33 g of biomass and 1 g of catalyst. The reaction was carried out at 60 °C with shaking at 200 rpm for 12 h, and then the solution was subjected to centrifugation at 8000 × g for 10 min to separate the enzyme linked nanoparticle from the hydrolysate. The upper liquid phase was used to determine the hydrolysed carbohydrate fraction, slurry like middle phase consisted of unhydrolysed biomass which was used for crystallinity index analysis, and the bottom thick solid phase was used to determine the immobilized enzyme and activity recoveries [22]. 2.4.
pH and thermal stability of immobilized enzyme during hydrolysis
The nanoparticle-linked cellulase prepared under optimised condition was added into a 125 mL Erlenmeyer flask containing 50 mL of water and 3.33 g biomass. The effects of pH (4.5, 7
5.0, 5.5, 6.0 and 6.5) and temperature (50, 55, 60, 65 and 70 °C) were analysed. After 12 h incubation with shaking at 200 rpm, the samples were centrifuged at 8000 × g for 10 min to separate the enzyme linked nanoparticle from the hydrolysate. The upper liquid phase was used to determine the glucose concentration and desorbed enzymes and the bottom thick solid phase was used to determine the immobilized enzyme and activity recoveries [22]. The percentage yield of glucose was calculated as the % conversion of available cellulose to glucose assuming the theoretical yield of 1.111 g/g cellulose [2]. 2.5.
Recycling of MgO-Fe3O4 linked enzyme catalyst and scale up studies
For recycling studies, 1 g of nanoparticle linked enzyme was used in 125 mL Erlenmeyer flask containing 50 mL of water and 3.33 g of biomass. Each hydrolysis cycle was run for 12 h at 60 °C and pH 5.5. To analyse the magnetic separation of immobilized enzyme, the immobilized enzymes were recovered using magnetic separation and the number of recycling was repeated up to seven cycles [22]. Briefly, 50 mL of hydrolysis medium containing immobilized enzyme was placed on magnetic separation stand (Lifesep 50SX) for 20 min to deposit the immobilized enzyme at the back wall. The supernatant was used for glucose yield whereas the deposited particles (immobilized enzyme) were washed twice with deionized water under magnetic separation. For scale up studies, the volume was increased to 1 L (1 L water, 66.6 g biomass and 20 g catalyst) and the rest were maintained under optimized conditions. 2.6.
Analytical methods
2.6.1. Physical characterization of catalyst and biomass The structure, size, shape and elemental composition of the MgO-Fe3O4 were characterized using field emission scanning electron microscope (FESEM) from CARL ZEISS, Germany and energy dispersive spectroscope (EDS) from Oxford instruments, United Kingdom. 8
Binding of enzyme onto nanoparticle was characterized by Fourier-transform infrared spectroscopy (FTIR) analyser (Perkin-Elmer FTIR spectrophotometer 2000 series) with the detector range at 4 cm-1 resolution and 25 scans per sample. The spectra were recorded between 4000 and 500 cm-1 [20]. The crystalline nature of the biomass was analysed using a Rigaku RINT-TTR3 X-ray diffractometer (Rigaku Co., Tokyo, Japan). The nickel-filtered CuKα radiation (k = 0.1542 nm) was applied at 50 kV and 30 mA. Samples were scanned over the range of 2h = 10–50 and the crystallinity index (CrI) was determined using Eqn. (1): [20]. CrI
I Crystalline I Amorphous I Crystalline
100 %
(1)
where, Icrystaline = intensity at 22°and Iamorphous= intensityat 18.8°. 2.6.2. Chemical characterization of hydrolysate The sugars such as glucose, galactose, mannose, xylose and arabinose were quantified using HPLC system (Shimadzu, Japan) equipped with refractive index detector (RID 10A, Shimadzu, Japan). The sugars were separated in Phenomenex, Rezex ROA-Organic acid column (150 × 7.8 mm) by using 5 mM H2SO4 as a mobile phase at a flow rate of 0.6 mL/min [23]. Polysaccharide contents in biomass were determined by acid hydrolysis method and the theoretical factors 1.111 and 1.136 were used for hexose and pentose sugars, respectively [2,24]. Total phenolics in biomass were determined by Folin-Ciocalteu method, using vanillin as a standard [25]. Protein content was estimated according to the method of Bradford using bovine serum albumin as standard [26]. The degree of oxidation of xylan was estimated using the method described by Zhao and Heindel [27]. Briefly, 0.1 g xylan aldehyde was suspended in 25 mL of 250 mM hydroxylamine hydrochloride solution, and then the pH was adjusted to 4.0 with 100 mM NaOH. The solution was stirred at room 9
temperature for 2 h, and then the suspension was titrated back to pH 4.0 with 100 mM NaOH. The degree of oxidation was calculated from the amount of NaOH consumed. 2.6.3. Enzyme characterization The crude enzyme extract, enzyme cocktail and biomass hydrolysate solution were used to determine filter paper (FPase), carboxymethylcellulase (CMCase), β-glucosidase and xylanase activities. One unit of activity (U) is defined as the amount (μmol) of product formed per minute under standard assay conditions [28]. The Fpase activity in immobilized enzyme was determined by adding 50 mg of metal nanoparticle linked enzyme and 50 mg of filter paper into 1.5 mL of 0.05 M sodium citrate buffer (pH 4.8) followed by incubation at 50 °C for 60 min. Dinitrosalicylic acid reagent was then added to the reaction mixture and the supernatant obtained after centrifugation (8,000 x g, 10 min) was used to determine the activity colorimetrically at 540 nm. The effect of nanoparticle on secondary structure of cellulase was analysed using circular dichroism (CD) spectroscopy. For every CD spectroscopic analysis, purified individual endo-glucanase, cellobiohydrolase and βglucosidase (0.1 mg protein/mL) was bounded with nanoparticle under optimized condition and was used for hydrolysis as mentioned in hydrolysis section [23,29]. The Far-UV CD spectra were recorded with spectropolarimeter (Jasco J-810, Japan) at 190–250 nm wavelength regions. The average values of three scans were used as a mean residue ellipticity [θ] (deg cm2 dmol−1) and the scans were analysed for secondary structure prediction using the K2D3 Algorithm [29]. 2.7.
Statistical analysis
All experiments were performed in triplicate and the average values are reported. The average and standard deviation values were calculated using the respective functions 10
(AVERAGE, STDEV) available in Microsoft Excel and the maximum difference among the three values was less than 5% of the mean. Statistical significances between the parameter levels were evaluated using Duncan's multiple range test at an α level of 0.05 (P<0.05). 3.
Results and Discussion
3.1.
Composition of raw biomass
The oven dried Chlorella sp. CYB2 biomass contained mass fraction of 38.32% total carbohydrates, in which the sugar components are glucose, galactose, mannose, arabinose and xylose (Table 1). Cellulose (33.6%) was the major component of total carbohydrates (Table 1). The lipids (17.4%), proteins (25.4%), phenolics (4.0%), ash (2.4%) and moisture (3.6%) were also present in biomass. The high content of carbohydrate fraction makes Chlorella sp. CYB2 biomass as a potential feedstock for fermentable sugar production. The composition of Chlorella sp. CYB2 observed in this study meets the feedstock criteria for biofuels production reported in previous studies [6,7]. 3.2. Cross linking of cellulase onto xylan aldehyde coated MgO-Fe3O4 In this study, the mixed oxides of Fe3O4 and MgO were developed as a support material for cellulase. As can be seen in Fig. 1a, the increase in MgO concentration from 100 to 400 mg/g increased the immobilization yield from 40 to 60.7%, beyond that it was saturated. This might be due to the disruption of ionic interaction between Fe3O4, MgO and enzyme caused by excessive addition of MgO. The hydrolysability was increased from 44.7 to 63.3% with an increase in MgO to Fe3O4 ratio, which indicates the significant influence of MgO on enzyme reactivity. According to Blanch [2], metal oxides develop cationic or anionic surface charge and alter the deprotonated carboxyl and phenolic site present in cellulosic biomass, which selectively disturb the cellulase inhibitors present in the biomass [2]. As shown in Fig. 1a, the 11
values for activity recovery after hydrolysis were varied from 76.7 to 96.3%, which indicate that the presence of MgO in nanoparticle improved the recovery of Fpase, CMCase and βglucosidase activities (Table S1). Overall, the immobilization yield and activity recovery influenced the hydrolysability of Chlorella sp. CYB2 biomass. The aldehyde groups of organic molecules have been utilized for cross-linking between the supporting material and enzyme molecules [15]. In this study, the BWX aldehyde has been utilized to improve the immobilization yield, which actually carries the aldehyde content of about 2.1 mM/g. As presented in Fig.1b, the increase in xylan aldehyde content increased the immobilization yield and hydrolysability until 60 mg/g, thereafter no significant improvement was observed. The results clearly indicate that the increase of xylan aldehyde content promotes more binding of enzyme onto nanoparticles via the increased linkage between aldehyde groups on surface of nanoparticles and amino groups of enzyme molecules. Similar results were obtained by Namdeo and Bajpai [30] when they used two different concentrations of oxidised cellulose for immobilization of α-amylase onto magnetic nanoparticles. The higher concentration of aldehyde was found to be a better option for the attachment of enzyme in immobilization. On the other hand, the activity recovery after hydrolysis was constantly increased with an increase in xylan aldehyde concentration and it reached the maximum activity recovery of 100% at 100 mg/g xylan aldehyde. The improvement of cellulase activity recovery in the presence of aldehyde group has been reported by Alahakoon et al. [31] with the immobilization yield of 52.4%. In the present study, the maximum immobilization yield of 88% and glucose yield of 76% were obtained at xylan aldehyde concentration of 60 mg/g, which indicates the potential of xylan aldehyde for use in immobilizing the enzymes.
12
The effects of enzyme concentration on immobilization yield, hydrolysability and activity recovery were investigated and the results are shown in Fig. 1c. Although, the increase in enzyme concentration slightly decreased the immobilization yield, it increased the total enzyme concentration bound to metal nanoparticles (Table S1). The maximum immobilization yield of 88.2% was observed at 150 mg/g of xylan aldehyde coated metal nanoparticles. The enzyme to nanoparticle ratio from 50 to 150 mg/g increased the hydrolysability from 56% to 90% and thereafter it reached the saturation. The saturation of enzyme binding to aldehyde surface was also reported in horseradish peroxidase [32] suggesting that the binding saturation is common irrespective of proteins or aldehyde source used. Besides, the activity recovery after hydrolysis was also found to increase with an increase in enzyme concentration from 50 to 100 mg/g and after which no significant difference in further increase of enzyme concentration (Fig. 1c). 3.3.
Physical characteristics of nanoparticle linked to enzyme
The morphology and composition of Fe3O4, MgO-Fe3O4 and enzyme linked MgO-Fe3O4 were analysed by FESEM-EDS and the results are shown in Figs. 2a – 2i. The spectrum results (Figs. 2a, 2d and 2g) show the presence of metal components with minor impurities. As presented in Figs 2b, 2e and 2h, the MgO-Fe3O4 particles were oval in shape and the average size was increased to ≈34 nm in diameter from ≈ 0.18 nm. After binding with the enzyme complex, the particles remained discrete and had a diameter of ≈34 nm. Similar results were reported by Jordan et al. [22] for immobilization of cellulase with magnetic nanoparticles. Figures 2c, 2f and 2i represent the weight percentage of individual components. As presented in Fig. 2i, the composition of metal oxides in enzyme linked MgOFe3O4 was similar to the composition of initial starting materials. The increase in carbon and oxygen contents in enzyme linked metal oxide (Fig. 2i) compared to that in metal oxide (Fig. 13
2f) indicates the presence of enzymes and xylan aldehyde in metal oxide nanoparticles. Although the carbon content was much lower than that of metal, the carbon content was equivalent with the content of protein and xylan aldehyde added. The binding of cellulase onto nanoparticle assisted by xylan aldehyde was confirmed by FTIR spectroscopy analysis. The FTIR spectra in Fig. 3 represent the spectra of Fe3O4, MgOFe3O4 -xylan aldehyde and cellulase-xylan aldehyde-MgO-Fe3O4. A stretch in the peak at 648 cm-1 indicates the Fe-O bond and the major peaks at 512, 586 and 671 cm-1 indicate the presence of stretching vibration of Mg-O, which demonstrated that MgO was successfully loaded with Fe3O4.The strong characteristic peaks at 1497 cm−1 and 1636 cm−1 were related to the stretching of C-O and C = O groups, respectively). By comparing the spectra of the xylan aldehyde coated MgO-Fe3O4 and Fe3O4 nanoparticles, it is observed that the bond around 3356 cm-1 is more pronounced and broader in xylan aldehyde coated than in the Fe3O4, indicating the presence of xylan aldehyde shell. The peak at 1544 cm-1 and the broadening of 1237 cm-1 peak are likely due to the formation of amide bond resulting from the linking of cellulase onto xylan aldehyde coated MgO-Fe3O4 [22]. A peak at 1041 cm−1 in enzyme linked MgO-Fe3O4 resembles the peak of cellulase molecule [11]. This characteristic shift in the frequency of cellulase-bound nanoparticles from the xylan aldehyde coated MgOFe3O4 is suggestive of covalent bonding of cellulase onto the nanoparticle [11]. Hence the results of FTIR confirmed successful mediation of xylan aldehyde on linking MgO-Fe3O4 with enzyme molecules. The secondary structural changes of individual cellulase components (endoglucanase, cellobiohydrolase and β–glucosidase) after linking MgO-Fe3O4 with enzyme were analysed using circular dichroism spectroscopy and the results are shown in Figs. 4a and 4b. The ellipticities were varied in all enzyme molecules, which indicate the alteration in molecular 14
shape of the cellulase (Fig. 4a). The free enzymes showed broad ellipticity whereas this was decreased with the enzymes bound to nanoparticles. The secondary structure of endoglucanase, cellobiohydrolase and β-glucosidase contains 27, 31 and 44% of α-helix and 22, 17, 14% of β-sheet, respectively (Fig. 4b). The results on secondary structure of nanoparticle linked enzyme revealed that the α-helix values were increased in cellobiohydrolase and β-glucosidase, while it was constant in endoglucanase. The β-sheet values were also increased in cellobiohydrolase and β-glucosidase, while it was decreased in endoglucanase. Mishra and Sardar [33] analysed the secondary structural changes of cellulase immobilized on silver and gold nanoparticles. The authors proved that the retaining of 85% α-helix structure is a crucial factor for retaining its activity. In the present study, the α-helix values were increased in cellobiohydrolase and β-glucosidase correlating well with the high activity recovery of CMCase and β-glucosidase at 95.2 and 98.6%, respectively, which were in agreement with previous studies [34,35]. 3.4.
Functional properties of nanoparticles and cellulase
In order to distinguish the role of metal oxides and xylan aldehyde in immobilization yield, hydrolysis and activity recovery after hydrolysis, each component was employed for the enzyme linked nanoparticle preparation and hydrolysis of microalgal biomass (Table 2). The immobilization yield of nanoparticle to the enzyme was less in Fe3O4 (59%) compared to MgO (71%), whereas the mixed metal oxides showed higher efficiency of 78%. Xylan aldehyde coated nanoparticles showed highest immobilization yield of 91.5%, which indicates the significance of xylan aldehyde coating in the linking of enzyme molecule to metal oxide nanoparticles. To determine the structural destabilisation of biomass, individual biomass obtained from different treatments (Fe3O4, MgO, MgO-Fe3O4, enzyme, and metal nanoparticle linked enzyme) were analysed using XRD (Fig. S1) and the values are 15
represented in Table 2. The crystallinity index of biomass in all treatments were reduced (22.1% - 28.3%) when compared to the raw biomass (30.1%, data not shown), while the highest reduction of crystallinty (22.1%) was observed in metal nanoparticle linked enzymatic hydrolysis. Hence, the reduced crystallinity facilitates the accessibility of enzymes to the substrate during the hydrolysis of cellulose. This contention is well supported by the glucose yield of 91.4% from the nanoparticle linked enzyme compared to the glucose yield of 77.2% from the free enzyme. Oligosaccharides were also detected after treatment of cellulose with Fe3O4, MgO and MgO-Fe3O4 giving the oligosaccharide yield of 1.2, 0.9 and 1.5 g/L, respectively (no oligosaccharide was detected in the control without nanoparticle). The results indicate that the metal oxides themselves have the hydrolysing ability to digest the cellulose, in which the oligosaccharides remained in different oligomers. Xie et al. [17] used MgO as a solid alkali for structural destabilization of cellulose and reported the significant reduction in degree of polymerization during hydrolysis. Similarly, Kobayashi et al. [16] observed the degradation of 1–4 linkage of cellulose when ruthenium was used as a catalyst. From the results, it can be confirmed that each individual component plays an important role in the hydrolysis of cellulose. The activity recoveries of three enzymes (Fpase, CMCase and β-glucosidase) after different treatments (Fe3O4, MgO, MgO-Fe3O4, enzyme, and metal nanoparticle linked enzyme) were analysed to determine the stability of the enzymes. As can be seen in Table 2, the activity recoveries after hydrolysis were higher in MgO-Fe3O4 than in free enzyme, which indicates the stability of enzyme was improved when linking onto metal nanoparticle. However, the activity recovery was highest in enzyme linked to xylan aldehyde coated MgO-Fe3O4, which indicates that the properties of a linking molecule can also contribute to the stability of the enzyme.
16
3.5.
Effect of pH and temperature on hydrolysis and stability of nanoparticle linked
enzyme For cellulases, the optimum pH 4.8 has been used in most studies for experimental purposes [28,31,36,37]. In the present study, the effects of initial pH on hydrolysis, activity recovery and desorption were investigated using the pH from 4.5 – 6.5 and the results obtained are shown in Fig. 5a. The hydrolysability was increased with an increase in pH and the maximum hydrolysability observed was 89% at pH 5.5, thereafter it was declined possibly due to the deactivation of enzyme at higher pH. The maximum activity recovery in terms of Fpase was 98% at pH 5.5, which is similarly observed for CMCase and β-glucosidase activities (Table S2). To analyse the amount of retaining protein content, desorption was analysed. The protein desorption was decreased with an increase in pH and reached the minimum desorption of 3% at pH 5.5, after that it was increased. The results confirmed that the increase in pH is likely to leach the protein from nanoparticle. Gokhale and Lee [36] immobilized cellulase and observed the swelling of cellulase which started at pH 7.0 and continued till pH 10.5. The authors also concluded that the biocatalytic potential of cellulase in these pH ranges was highly doubtful [36]. In the present study, desorption was below 7.3% in all tested pHs and the maximum activity recovery of 97% was observed at pH 5.5. Considering the protein and activity recoveries, pH 5.5 was selected as an optimum. The linking of the enzyme to a supporting material can increase the thermal stability by stabilizing the weak ionic forces and hydrogen bonds and thus increasing the range of operating temperatures. However, the denaturation of enzyme and reduced activity is also possible with higher temperatures. The changes in hydrolysability, activity recovery and protein desorption were analysed at different temperatures and the results are depicted in Fig. 5b. The hydrolysability was increased with an increase in temperature and the maximum 17
hydrolysability observed was 91% at 60 °C, thereafter it was reduced to 79% at 70 °C. The protein desorption was not significantly affected at all temperatures until 65 °C, thus confirming the possibilities of recovering protein even at high temperature. The activity recovery was slightly altered with an increase in temperature and the maximum activity recovery was observed at 60 °C. As the thermal stability increases, the cellulase can act at higher temperature resulting in an easy solubilization of cellulosic biomass. 3.6. Recycling of nanoparticle linked enzyme molecule The reuse of recovered nanoparticle linked enzyme for hydrolysis is a crucial factor for economic efficiency of the hydrolysis with enzymes, since enzymes are more expensive than chemical agents. Immobilized enzymes were separated using magnetic separation and were subjected to seven cycles of hydrolysis to analyse their efficiency after every cycle. As presented in Fig. 6, the activity recovery in immobilized enzyme was slightly decreased after each cycle of hydrolysis. After seven cycles, about 84.5% activity was retained compared to about 97% at the start of the experiments. The decrease in activity might be due to both desorption and denaturation of enzymes. Similar reusability studies on immobilized cellulase have been reported. Gokhale et al [37] immobilized cellulase directly onto magnetic nanoparticles; however, the enzyme retained about 55% of original activity after four cycles of reuse. Alahakoon et al. [31] compared the use of amine and aldehyde functionalized Fe3O4 magnetic nanoparticles for cellulase immobilization and reported that aldehyde functionalized nanoparticle retained higher activity (76.5–87%) than amine functionalized nanopartilce (70.1–76.4%) after six cycles. In comparison, the present study achieved higher activity recovery than the research studies reported earlier. The improved activity recovery is mainly attributed to the flexible interaction between metal nanoparticle and enzyme molecule assisted by xylan aldehyde. It is noted that both the catalyst recovery and the glucose yield were relatively unaffected even after seven cycles of hydrolysis. 18
3.7.
Scale up studies on hydrolysis
The scale up studies were carried out by increasing the reaction volume to 1 L under optimized condition. The scale up studies up to 1 L showed almost 90% of glucose yield which confirms the application of nanoparticle linked enzymatic catalyst at a large scale. Hydrolysis of 100 g of algal biomass resulted in 22.5 g of glucose whereas the unhydrolysed carbohydrate, lipid and protein contents were recovered as a solid biomass. Overall, the glucose production of 0.336 g/g of original biomass was observed after saccharification and 0.034 g/g of unhydrolysed cellulose was retained in solid content. The quantity of immobilized enzyme added was 30 g per 100 g of biomass; however, the optimization of catalyst loading can reduce the catalyst requirement in commercialization. In general, the reaction of solid biocatalyst with solid substrate is low due to the poor accessibility of the enzyme to the substrate. In most of the previous studies the plant biomass was pretreated before hydrolysis using immobilized enzymes. The pretreatment renders the substrate present in the biomass more readily accessible to the enzyme. Kumakura [38] immobilized cellulase onto aluminium oxide beads and observed 53% hydrolysis of cellulose component of chaff, pretreated by electron beam irradiation. Similarly, the immobilization of cellulase onto cyclodextrin conjugated magnetic particle improved hydrolytic efficiency on rice straw after pretreatment by grinding into powder form [39]. Abraham et al. [11] immobilized cellulase for the hydrolysis of hump hurd biomass pretreated with NaOH and reported maximum hydrolysis of 93% in immobilized enzyme which is comparatively higher than free enzyme (89%). However, in this study most of the algal cellulose were converted to glucose, which indicates the potential of the developed immobilization method in algal biomass utilization. Unlike plant biomass, the algal biomass becomes more susceptible for hydrolysis, which is partly due to the reduced particle size and the amorphous nature of algal biomass compared 19
to plant biomass. However, a separate pretreatment process and a suitable immobilization method should be evaluated when using lignocellulosic plant biomass. The efficient recovery of immobilized enzyme and an improvement of hydrolysis make the process attractive with high potential for industrial utilization of algal biomass for fermentable sugar production.
4. Conclusion The development of a viable bioconversion process for fermentable sugar production is the major challenge in cellulosic ethanol production process. This study demonstrated that MgO improved the hydrolysability of biomass, whereas the Fe3O4 facilitated the separation of catalyst. Addition of xylan aldehyde improved the enzyme binding to the metal nanoparticle, without reducing the hydrolytic performance. Furthermore, the scale up study provides more insight into its commercialization potential. Acknowledgements R.V. is thankful to the Graduate School and Faculty of Science, Chulalongkorn University (CU), for post-doctoral fellowship from Rachadaphiseksomphot Endowment Fund. A.I. acknowledges the research grant from CU on the Frontier Research Energy Cluster (CU-59048-EN). Appendices Immobilization yield was obtained from immobilization experiments and was calculated from the following equation (Eq. A.1) Im mobilizati on yield
( I P LP ) 100 IP
(A.1)
where, LP is the concentration of protein observed in liquid (mg/50 mL), IP is the concentration of protein added (mg/50 mL).
20
Hydrolysability was determined after hydrolysis experiment and was calculated from the following equation (Eq. A.2) Glu cos e yield
Qglc (Qcellulose 1.111)
100
(A.2)
where, Qglc is the concentration of glucose observed in hydrolysate (g/50 mL), Qcellulose is the concentration of cellulose (quantity of biomass × 0.336) added (g/50mL), 1.111 is the theoretical factor. Activity recovery was determined after every hydrolysis experiment and was calculated from the following equation (Eq. A.3) Activity re cov ery
Factivity I ativity
100
(A.3)
where, Factivity is the total activity obtained after hydrolysis (U/ mL), Iactivity is the initial activity in immobilized enzyme (U/ mL). Desorption of protein after hydrolysis was calculated from the following equation (Eq. A.4)
Desorption
I protein Fprotein I protein
100
(A.4)
where, Iprotein is the concentration of nanoparticle linked protein added (mg/50 mL), Fprotein is the concentration of protein recovered along with nanoparticle (mg/50 mL). References [1] D.O. Hall, Biomass for energy – a worldwide view, In: E. Campos-Lopez (Ed), Renewable resources a systematic approach, Academic Press, USA, 1980, pp. 287–305. [2] H.W. Blanch, Bioprocessing for biofuels, Curr. Opin. Biotech. 23 (2012) 390–395. [3] P. Savakis, K.J. Hellingwerf, Engineering cyanobacteria for direct biofuel production from CO2, Curr. Opin. Biotechnol. 33 (2015) 8–14. [4] X. Zeng, M.K. Danquah, X.D. Chen, Y. Lu, Microalgae bioengineering: From CO2 fixation to biofuel production, Renew. Sust. Energ. 15 (2011) 3252–3260. 21
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doi.org/10.1155/2015/409103.
Figure Captions Fig. 1 Factors affecting immobilization yield, activity recovery and glucose yield (a) MgO to Fe3O4 ratio, (b) xylan aldehyde concentration, (c) protein concentration. Symbols: ■immobilization yield ■-activity recovery, □- glucose yield. Different letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05. 25
Fig. 2 FESEM-EDS analysis of Fe3O4 (a-spectrum, b-microscopic image, c-weight percentage), xylan aldehyde coated MgO-Fe3O4 (d-spectrum, e-microscopic image, f-weight percentage), xylan aldehyde coated MgO-Fe3O4 linked to enzyme (g-spectrum, h-microscopic image, i-weight percentage). Fig. 3 Fourier transform infrared spectra of (a) Fe3O4 (b) xylan aldehyde coated MgO-Fe3O4, (c) enzyme linked xylan aldehyde coated MgO-Fe3O4. Fig. 4 (a) Circular dichroism spectrum (symbols: (1) EG control (2) EG- MgO-Fe3O4 (3) CBH
control (4) CBH- MgO-Fe3O4 (5) BGL-control (6) BGL- MgO-Fe3O4) and (b) Percentage αhelix and β-sheet values (symbols: ■-α helix and □-β sheet) of endoglucanase I (EG: 46 kDa), cellobiohydrolase I (CBH: 56.2 kDa) and β-glucosidase (BGL: 75 kDa) for control (without MgO-Fe3O4) and MgO-Fe3O4 linked enzyme. Different letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05. Fig. 5 Effect of temperature on glucose yield, residual activity and desorption of protein. Symbols: ■-glucose yield, ■-activity recovery, □-desorption. Different letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05. Fig. 6 Recycling of metal nanoparticle linked enzyme and glucose yield after each cycle of hydrolysis. Symbols: ■-catalyst recovery, ■- glucose yield, □- activity recovery. Control represents the analysis at the start of the experiments. Different letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05.
26
Immobilization yield (%); activity recovery (%); glucose yield (%)
(a)
120 100
d
e
80
b
60 40
d
e
d
a
b
a
a
a
c
20 0 Control
100
200 300 400 MgO to Fe3O4 ratio (mg/g)
(b) 120
Immobilization yield (%); activity recovery (%); glucose yield (%)
c
d
a
a,b
b
c
100
b
80
c
c
a a
a
60 40 20
0 20
40 60 80 Xylan aldehyde concentration (mg/g)
(c) 120 Immobilization yield (%); activity recovery (%); glucose yield (%)
a
a
b
a
b
a b,c
c
c
500
100
a
a
c
80
a
a
a
a,b
b
100
a
a
a
b
b c
c
60 40 20 0 50
100 150 200 Protein concentration (mg/g)
250
Fig. 1
27
(c) 80 Weight percentage (%)
(b)
(a)
Weight percentage (%)
80 60 40
20
60 40
20
0 C
O
30 µm
(d)
S Fe Components Electron Image 1
(e)
Weight percentage (%)
(f)
Weight percentage (%)
80 60 40
20 0 C
O
30 µm
S Fe Components Electron Image 1
O
S Fe Components
Mg
C
O
S Fe Components
Mg
C
O
S Fe Components
Mg
80
60 40
20 0
(i) Weight percentage (%)
80
Weight percentage (%)
80 60 40
20 0 C
KeV
C
Mg
(h)
(g)
0
Mg
30 µm
O
S Fe Components Electron Image 1
60 40
20
Mg
0
Fig. 2
28
Transmittance Transmittance (not (not for scale)
(a) (a) (b) (b) (c)
1636 1497 1544 1237 1041
3356
671 512 586
4000 4000
3500 3500
3000 3000
2500 2000 1500 2500 2000 1500 Wave number (cm-1) Wave number (cm-1)
1000 1000
500 500
Fig. 3
29
(a)
10
(6) (5)
8
(4) (3)
[θ] (103 degcm-2 dmol -1)
6 4 2
(2) (1)
0 -2 -4 -6 -8 190
200
210 220 Wavelength (nm)
230
240
(b) 50
a
a
45
α-helix and β-sheet values (%)
40 35 30 25 20
b
c d
d a
b c
15
c
e
d
10 5 0 EG EG CBH CBH BGL BGL (Control) (MgO-Fe (MgO-3O4) (Control) (MgO-Fe (MgO(Control) (MgOO ) (MgO-Fe 3 4 3O4) Fe3O4) Fe3O4) Fe3O4)
Enzymes
Enzymes
Fig. 4
30
Glucose yield (%); activity recovery (%); desorption (%)
(a) 120 a
100
b
c 80
a
a
a
b b
c
c
60 40 20
a
a
a
a
a
0 4.5
5.5
6.0
6.5
a
a
pH 120
Glucose yield (%); activity recovery (%); desorption (%)
(b)
5.0
a
a
100
a a
b
c
b
c
80 60
40 20
b
b
b
a
b
0 50
55
60 65 Temperature (°C)
70
Fig. 5
31
Immobilized enzyme recovery (%); glucose yield (%); activity recovery (%)
120
100
a aa
1
a
aa
2
a a a
a a a
a a a
3 4 5 Number of cycles
a
a
6
a
a a
b
80
60
40
20
0
7
Fig. 6
32
List of Tables Table 1 Composition of Chlorella sp. CYB2 Components
Concentration (%, w/w)
Cellulose
33.6±0.78
Galactan
1.8±0.49
Manosan
0.9±0.38
Arabinan
1.1±0.32
Xylan
1.0±0.41
Lipids
17.3±0.70
Protein
25.3±0.67
Phenolics
4.0±0.89
Ash
2.4±0.55
Moisture
3.6±0.78
Other
9±1.32
33
Table 2 Comparison of individual and immobilized catalyst on immobilization yield, activity recovery, and hydrolysis of Chlorella sp. CYB2 cellulose content. Activity recovery b Immobiliz Compon ation yield a ent
FPase
CMCa se
βglucosi dase
(g/g, %)
Crystall Hydrolytic efficiencyc inty index glucos cellobi oligo- glucose sacchar of e ose yield ide biomas (g/L) (g/L) (%) (g/L) s (%)
Fe3O4
59.4±2.1d
76.0± 2.5c
45.8± 4.4d
74.7±1. 5d
28.3±1. 4a
0.1±0. 0c
0.7±0. 1b
1.2±0. 1a
1.0±0. 1c
MgO
71.5±4.4c
91.1± 0.6b
99.4± 4.3a
92.8±1. 4b
26.4±2. 2b
0.1±0. 1c
0.5±0. 2b
0.9±0. 3a,b
1.0±0. 3c
MgOFe3O4
77.6±3.9b
78.1± 1.8c
62.8± 2.6b
85.3±1. 7c
24.8±1. 6c
0.2±0. 0c
0.9±0. 2b
1.5±0. 3a
1.4±0. 2c
Enzy me
na
76.2± 4.6c
59.7± 3.2c
75.2±2. 2d
27.6±0. 7a,b
14.5± 0.4b
1.3±0. 4a
0.3±0. 4b
77.2± 2.1b
Enzy meXylan aldehy deMgOFe3O4
91.5±3.5a
97.6± 1.9a
95.2± 1.1a
98.6±1. 2a
22.1±2. 1d
17.1± 0.3a
0.8±0. 3b
0.2±0. 1b
91.4± 1.5a
na-not applicable. a immobilization yield was analysed by adding the protein concentration of 150 mg/g metal oxide; b activity recovery was determined after hydrolysis; c hydrolytic efficiency was carried out with individual component as mentioned in first row. The superscript letters on rows indicate the significant difference and the same letter indicates no significant difference according to Duncan's multiple range test at P ≤ 0.05.
34