Investigation on the Immobilization of Carbonic Anhydrase and the

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Investigation on the Immobilization of Carbonic Anhydrase and the Catalytic Absorption of CO2 Juan Li, Xincheng Zhou, Lin Zhang, Huajuan Di, Hao Wu, and Linjun Yang Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.6b02652 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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Energy & Fuels

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Investigation on the Immobilization of Carbonic Anhydrase and the

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Catalytic Absorption of CO2

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Juan Li, 1 Xincheng Zhou, 1 Lin Zhang, 1 Huajuan Di, 1 Hao Wu, 1 Linjun Yang*, 2, 11

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1

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Education, School of Energy and Environment, Southeast University, Nanjing 210096,

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Jiangsu Province, China

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2

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Province, China University of Mining and Technology, Xuzhou 221008, Jiangsu

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Province, China

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Key Laboratory of Energy Thermal Conversion and Control of Ministry of

Key Laboratory of Coal-based CO2 Capture and Geological Storage, Jiangsu

ABSTRACT

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The optimum conditions, such as pH, immobilization time, temperature, and

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enzyme dose for carbonic anhydrase (CA) immobilized on the polyester polyurethane

13

prepolymer 80 were investigated. Simultaneously, the performances (e.g., the pH,

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thermal, operational and storage stability) of the immobilized CA and the free CA

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were compared. The results revealed that the optimum conditions, such as pH value of

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Tris-HCl buffer, immobilization time, temperature and enzyme dose of the

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immobilized CA were 7.5, 24 h, 35 °C, and 1 mL enzyme solution (0.1 mg/mL)/0.1 g

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polyester 80, respectively. Moreover, the pH, operational, thermal and storage

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stability of the immobilized CA was higher than that of the free CA. On this basis, the

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experiment was conducted to immobilize CA to absorb CO2. The results indicated that

*

Corresponding author. E-mail: [email protected].

1

ACS Paragon Plus Environment

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the CO2 concentration, the liquid-gas ratio and the gas-flow rate may affect the

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properties of CO2 absorption. The CO2 absorption efficiency dropped from 63% to

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42.7% with an increase in the CO2 concentration from 9% to 15.4%. By increasing

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the gas-flow rate, the CO2 absorption efficiency first increased and subsequently

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decreased. The absorption efficiency increased from 30.9% to 57.7% with an increase

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in the liquid-to-gas ratio from 26 L/m3 to 34 L/m3. The optimal operating conditions

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can accelerate the absorption rate of CO2 and reduce the usage of immobilized CA,

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which can reduce the cost of CO2 sequestration.

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Keywords: Carbonic anhydrase (CA); Immobilization; CO2 capture; Catalytic

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absorption; Absorption efficiency

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1. Introduction According to the International Energy Agency (IEA), the total emissions of CO2

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[1]

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was 32.3 billion tons in 2014 in the global

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caused by the greenhouse effect, CO2 capture and storage technologies gained more

35

and more attention from the international [2]. In recent years, many scholars have used

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a kind of zinc metalloenzyme—carbonic anhydrase（CA), to capture CO2 effectively [3,

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4, 5, 6]

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The reaction rate of CO2 hydration with CA is 109 times as much as the rate without

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CA. Besides, CA is a stable, safe and harmless catalyst

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advantage of CO2 capture with CA is the carbonation of CO2 under a low

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concentration.

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. Due to a variety of climate issues

. CA is the most effective catalyst to catalyze CO2 hydration and dehydration [7].

[8]

. The most significant

Nevertheless, due to the shortcomings of poor stability and the difficulty in 2


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recycling free CA, the study of enzyme immobilization is particularly important.

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Immobilization technology can be one of the best approaches to reuse the enzyme and

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to establish a cost-competitive route for the commercialization of the process

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After immobilization, the performances of immobilization enzyme can be

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significantly improved [11, 12]. Normal supports for enzyme immobilization are nanometer material

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[15]

[16, 17, 18]

.

[13, 14]

,

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chitosan

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methods of immobilization, such as adsorption, the covalent bonding method,

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embedding and the crosslinking method

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from 1977, when Ray et al. fixed CA onto a polyacrylamide gel via embedding and

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found that the resistance of immobilization enzyme to heat and the sulfonamide

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reaction were improved [21]. To date, there are many researches on CA immobilization.

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However, the researches on immobilized CA catalyzing the reaction of CO2 hydration

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in a reaction kettle are limited. Zhang et al. and the company Carbozyme arranged the

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immobilized CA in a hollow fiber membrane reactor to capture low-concentration

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CO2, which provided a potential method for the application of immobilized enzyme

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[22]

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enzyme activity, operational stability and engineering amplification

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placed immobilized enzyme onto porous ceramic in a vertical reactor to catalyze and

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absorb CO2, which offered a baseline application for the immobilized enzyme to

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capture CO2 from air or flue gas [23].

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, polyurethane foam and seaweed salt

[9, 10]

, etc. There are four main

[19, 20]

. Immobilization technology could date

. However, many problems still exist, such as membrane fouling, the loss of [22]

. Zhu et al.

In this study, CA was immobilized on the polyester polymer 80 via the covalent

3


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bonding method. In addition, the optimum conditions for CA, such as pH,

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immobilization time, temperature and enzyme dose, were evaluated. The pH, thermal,

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operational and storage stability of the free and immobilized CA were also compared.

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Moreover, the properties of the immobilized enzyme, as the packing of the absorbing

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tower, to absorb CO2 were studied.

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2. Material and methods

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2.1 Chemicals and reagents

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CA separated from bovine erythrocytes was purchased from Nanjing Duly

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Biotech Co. Ltd. (Nanjing, PRC). Tris (hydroxymethyl) amino-methane (tris),

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4-Nitrophenyl acetate (p-NPA) and Acetonitrile were all obtained from Sino-pharm

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Chemical Reagent Co. Ltd. (Shanghai, PRC). Polyester polyurethane prepolymer 80,

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which was the carrier to immobilize CA, was obtained from Zibo Hengjiu Plastic

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Technology Co. Ltd. (Zibo, PRC). CO2 (99.99 %) and N2（99.9%）were used to

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generate the simulated flue gas for the CO2 absorption test. All chemicals were of

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analytical grade.

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2.2 Immobilization of CA and stability

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2.2.1 Immobilization of CA

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A CA solution (0.1 mg/mL) was prepared by mixing with 50 mmol Tris-HCl

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(pH=8.0), and CA was immobilized onto the polyester polyurethane prepolymer 80

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via the covalent binding method. Firstly, 0.3 g polyester polyurethane prepolymer 80

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was introduced into a Tris-HCl buffer solution (20 mL, pH=8.0) with 3 mL of enzyme

86

solution (0.1 mg/mL) in a 50 mL centrifuge tube, laid in a shaking table at 35 °C and 4


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100 rpm continuing for 24 h, which was the optimum condition, and only single factor

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was changed in the following experiments. To find the optimum conditions, the

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parameters, such as pH (7.1, 7.5, 8.0, 8.5, 9.0), immobilization time (8, 12, 16, 20, 24,

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28, 30, 32 h), temperature (20, 25, 30, 35, 40, 45 °C) and enzyme dose (1, 2, 3, 4, 5

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mL), were studied.

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Next, the product was washed with a buffer solution (10 mL, pH=8) 3 times and

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dried at 25 °C until reaching a constant weight. Finally, the immobilized CA was

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stored at 4 °C for the subsequent usage.

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2.2.2 Immobilized and free CA activity assay

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The activity of CA was estimated using UV/Vis spectrophotometer and p-nitro [24]

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phenyl-acetate (p-NPA)

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of p-NPA to generate p-NP. Then, the enzyme activity was determined by measuring

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the concentration change of p-NP, whose concentration was defined by

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spectrophotometric measurement. The calculation formula of the enzyme activity is as

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follows:

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103

. In our experiment, CA catalyzed the hydrolysis reaction

U=

D × 103 × (OD348,enzyme − OD348,black ) × Vt e p − NP × VS × d

Note: D——Dilution multiple;

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OD348, enzyme——Absorbance of the solution at 348 nm with adding enzyme;

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OD348, blank——Absorbance of the solution at 348 nm without adding enzyme

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solution;

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Vt——Total reaction volume，mL;

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ep-NP——Molar extinction coefficient of p-NP at 348 nm; 5


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VS——Volume of enzyme solution，mL.

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Firstly, 3mL of the enzyme solution (0.1 mg/mL) were mixed with 6 mL of

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Tris-HCl solution and 1 mL p-NPA (3 mmol /L, dissolved in acetonitrile). After

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reacting for 5 minutes, the absorbance of the solution at 348 nm at 30 °C was noted.

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The blank experiment was defined by recording the absorbance of the solution at 348

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nm without adding the free CA.

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The measuring approach to the activity of the immobilized CA was similar to

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that of the free CA. Firstly, 9 mL of Tris-HCl solution and the appropriate amount of

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immobilized CA were mixed with 1 mL p-NPA. Next, after 5 minutes, the

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immobilized enzyme was removed by filtration, and the absorbance of the filtrate was

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measured at 348 nm. The blank experiment was defined by recording the absorbance

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of the solution with adding the same amount of carrier material at 348 nm.

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There are three main methods used to determine the efficiency of enzyme

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immobilization, namely, the immobilization yield, the immobilization efficiency and

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the activity recovery [20]. In our experiment, the enzyme activity was evaluated by the

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activity recovery and the relative enzyme activity

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immobilization yield multiplied by the immobilization efficiency. In addition, the

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relative enzyme recovery was acquired by comparing the enzyme activity with the

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maximum enzyme activity (regarded as 100%) in the same set of experiments. Two

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expressions were more intuitive to define the enzyme activity and could be written as:

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[25]

Activity recovery(%) = Yield(%) × Efficiency(%) =

. Activity recovery is the

observed activity × 100% staring activity

6


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Relative enzyme activity(%) =

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Enzyme activity at each condition × 100% Enzyme activity at optimum condition

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2.2.3 Stability of the immobilized and free CA

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2.2.3.1 pH stability

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A Tris-HCl buffer solution (50 mmol) at different pH values (7.1, 7.5, 8.0, 8.5

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and 9.0) was added to the free enzyme and the immobilized enzyme for 1 h.

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Subsequently, the enzyme activity at each pH value was tested, as described in

136

Section 2.2.2.

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2.2.3.2 Thermal stability

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The impact of temperature on the enzyme activity was tested by incubating the

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reaction mixture in Tris-HCl (50 mmol, pH=8) with a temperature range of 20 °C to

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60 °C for 1 h.

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2.2.3.3 Operational stability

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The enzyme operational stability is one of the significant technical parameters [26]

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for practical application

. The appropriate amount of the immobilized CA was

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added to the absorption tube, with the continuous introduction of gas and absorbing

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liquid. Under actual operating conditions, enzyme activity was measured after

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continuous reaction for 1 h. The procedure was repeated for 5 times to investigate the

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operational stability of the immobilized CA.

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2.2.3.4 Storage stability

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The immobilized and free CA were stored at room temperature and at 4 °C for 14

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days, respectively, and the relative enzyme activity was tested at intervals of every 2

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days. 7


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2.3 The catalytic absorption of CO2 with immobilized CA

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The schematic of the experimental apparatus for the CO2 catalytic absorption

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with the immobilized CA is shown in Fig. 1. The simulation test-bed was consisted of

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a gas preparation system, a CO2 absorption system, and an analyzing and testing

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system. The CO2 absorption system was composed of a packing tower and a spray

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device. The simulated gas generated by gas cylinders flowed through a mass flow

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controller (Seven-Star Electronics Co., Ltd.) into the packing tower from the bottom

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after blending in a static mixer. The absorbent was pumped into the absorbing tower

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from the feed tank through a rota-meter using a peristaltic pump (Longer-Pump®

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BT100-300M). Two gas-liquid phases were counter-current flow in the CO2

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absorption device.

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The body of the absorbing tower was made of organic glass, with a

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column diameter of 5 cm, a tower body height of 75 cm and a packing height of 3 cm.

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In particular, the gas inlet and the packing layer (supported by a plexiglass support

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plate) from the bottom of column were 10 cm and 25 cm, respectively. The CO2

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concentration at the inlet and outlet of the absorbing tower was measured using an

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on-line gas analyzer (GXH-3010E1) and a multi-component gas analyzer

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(Rosemount Analytical CAT 100).

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In the CO2 catalytic absorption device, different parameters, such as the CO2

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concentration, the gas-flow rate and the liquid-gas ratio, were studied to investigate

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the performance of CO2 absorption with the immobilized CA.

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3. Results and discussion 8


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3.1 CA immobilization studies

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The effects of operating conditions on the immobilized enzyme are presented in

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Fig. 2. As seen in Fig. 2(a), the maximum activity recovery of the immobilized

177

enzyme was observed at pH=7.5. With an increase in pH from 7.5 to 9.0, the activity

178

of the immobilized CA decreased, due to the ionized state of the amino-acid residue

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within the activity center of CA changing for different pH

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pH, CA and p-NPA were in the most appropriate ionization state. In addition, enzyme

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activity was being degraded and inhibited at an excessively high pH for long time.

[27]

. When at the optimum

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As shown in Fig. 2(b), the activity recovery of the immobilized CA increased

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first and then decreased with the increasing of immobilization time. With short

184

immobilization time, the reaction equilibrium was not reached. When the

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immobilization time reached 26 h, the activity recovery was the maximum. After then,

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with the further extension of immobilization time, the enzyme activity recovery rate

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showed a downward trend. A part of enzyme molecules leach from the carrier due to

188

stirring and the enzyme is being degraded after exposure to reaction system for a long

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period [28]. However, the enzyme activity recovery rate increased slowly after 24 h, so,

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considering the reaction time and energy consumption, 24 h is selected to be the

191

optimum immobilization time for further study.

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The impact of temperature on the activity of the immobilized CA is shown in Fig.

193

2(c). With the increase of temperature, when the immobilized enzyme reached the

194

optimum temperature, the enzyme activity reached the maximum value and the rate of

195

enzymatic reaction was the fastest. Above or below 35 °C, the rate of enzymatic

9


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reaction decreased, thus the activity of the enzyme decreased.

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In the enzyme immobilization experiments, the dose of the carrier was constant

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(0.3 g) and the effect of enzyme dose on the immobilized CA activity is shown in Fig.

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2(d). With an increase in the enzyme dose, the activity of the immobilized CA

200

increased and then decreased. When the amount of the enzyme solution (0.1 mg/mL)

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was 3 mL, i.e., 1 mL enzyme solution/0.1 g carrier, the immobilized enzyme activity

202

arrived at the maximum. With a lower amount of enzyme, the number of the enzyme

203

molecules was limited to combine to the carrier, so, with the increase of the amount of

204

enzyme, the activity recovery rate was higher. However, with further increase in the

205

amount of enzyme, the active sites of the carrier decreased, so enzyme molecule

206

needed to overcome more mass transfer resistance to arrive at the active sites of the

207

carrier, which lead to lower reaction rate. Moreover, the enzyme molecules were easy

208

to assemble; therefore it is not conducive for the enzyme molecules to extend to the

209

direction of the carrier with an excessive amount of enzyme, which made insufficient

210

contact with the carrier. In general, both of reasons resulted in the decreased enzyme

211

activity.

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In summary, the optimum conditions for CA immobilization on polyurethane

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polyester 80 were ascertained as follows: (a) pH value, 7.5; (b) immobilization time,

214

24 h; (c) immobilization temperature, 35 °C; (d) enzyme dose, 3 mL enzyme

215

solution/0.3 g carrier. Under these optimum conditions, the activity recovery of the

216

immobilized CA could reach 59.82%.

217

3.2 Optimum conditions of the immobilized CA

10


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3.2.1 Optimum pH value of the immobilized CA

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The immobilized CA (obtained under the optimum conditions) and free CA were

220

added into the different pH of buffered solution, such as 7.1, 7.5, 8.0, 8.5, and 9.0,

221

respectively. The relative activity of them was measured under room temperature for

222

20 min, and the results are shown in Fig. 3. The optimum pH of the immobilization

223

CA was higher than that of the free CA. The immobilized CA is weakly acidic, which

224

needed higher pH of buffer solution. Besides, the immobilization of enzyme improved

225

the spatial structure of the enzyme, so the immobilized CA had higher optimum pH.

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3.2.2 Optimum temperature of the immobilized CA

227

The immobilized and free CA were added into the different temperature of buffer

228

solution for 20 min, such as 20 °C, 25 °C, 30 °C, 35 °C, 45 °C, respectively. The

229

relative activity of them was measured. From Fig. 4, the optimum temperature of the

230

immobilized CA was different of that of the free CA, which was 30 °C, 35 °C,

231

respectively. After immobilization, the spatial structure of CA was improved,

232

therefore the immobilized CA had higher optimum temperature.

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3.3 Stability study of the immobilized and free CA

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3.3.1 pH stability

235

The selection of the enzyme and the material determines the change of the

236

optimum pH. This change is beneficial to understand the structure-function

237

relationship of the enzyme and to compare the activity of free and immobilized

238

enzyme as a function of pH

239

immobilized and free CA first increased and then decreased with an increase in pH

[29]

. As shown in Fig. 5(a), the relative activity of the

11


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value. The optimum pH of the free CA and the immobilized CA were 7.5 and 8.0,

241

respectively. Moreover, the pH stability of the immobilized enzyme was higher than

242

that of the free enzyme.

243

3.3.2 Thermal stability

244

The effect of temperature on the immobilized CA is shown in Fig. 5(b). The

245

activity of the immobilized CA increased up to 35 °C and then decreased with an

246

increase in temperature. The change trend of the free CA was similar to that of the

247

immobilized CA, and the optimum temperature for the free enzyme was 30 °C. The

248

relative activity of the immobilized CA still maintained 40.18% at 60 °C. However,

249

the relative activity of the free CA was only 14.25% under the same conditions.

250

According to the above results, the thermal stability of the immobilized CA was

251

significantly improved. After immobilization, the enzyme molecule had more points

252

to connect with the carrier, which not only prevented the enzyme molecule from

253

extensional deformation but also enhanced the CA resistance to temperature.

254

3.3.3 Operational stability Enzyme operational stability is a significant parameter for practical application

255 256

[23]

257

cannot be recovered and reused, their re-usability was not considered. Nevertheless,

258

the relative activity of the immobilized CA could maintain 58.71% after continuous

259

reaction for 5 times, which indicated that the immobilized CA had good operational

260

stability. The immobilized CA released the activity slowly and suppressed enzyme

261

degradation. After reaction, the CA on the carrier was generated again and absorbed

. Fig. 5(c) shows the re-usability of the immobilized CA. Because free enzyme

12


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on polyester polyurethane prepolymer 80, which indicated the recyclability of the

263

immobilized CA.

264

3.3.4 Storage stability

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Fig. 5(d) illustrates that the immobilized CA can still maintain 86.79% relative

266

activity at 4 °C for 14 d. However, the relative activity of the free CA was only

267

22.34% under the same conditions. At room temperature for 14 d, the relative

268

activities of the immobilized and free CA were 55.56% and 6.2%, respectively. All of

269

these results indicated that the enzyme immobilization could enhance its storage

270

stability and resistance to environmental change.

271

3.4 CO2 catalytic absorption with immobilized CA

272

3.4.1 CO2 catalytic absorption

273

CO2 (500 mL/min, 99.99%) was introduced into aqueous solution (pH=7.0), at the

274

same time the pH of the absorbing liquid with free CA (1mg/water 1L), the

275

immobilized CA and without CA were measured by the pH electrode of ion analyze,

276

respectively. From Fig. 6, at the beginning, the absorption liquid with free CA could

277

absorb CO2 faster and earlier reach CO2 saturation than that with the immobilized CA

278

and without CA, which suggested that free CA and immobilized CA could catalyze

279

CO2 hydration reaction effectively.

280

3.4.2 The effect of CO2 concentration

281

As shown in Fig. 7, the absorption efficiency decreased with an increase in the

282

CO2 concentration. When the CO2 concentration varied from 9% to 15.4%, the

283

absorption efficiency dropped from 63% to 42.7%. However, the absorption quality of

13


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CO2 increased from 75 mg/min to 197 mg/min, which illustrated that the immobilized

285

CA still had better CO2 absorption efficiency. CO2 was catalyzed to HCO3- by the

286

immobilized CA during entering the tower at the lowest filler of the tower. Then, with

287

the increase of CO2 concentration, the efficiency of CO2 absorption decreased, but the

288

absorption quality of CO2 increased. At the low concentration, CO2 absorption is more

289

overall. With the increase of CO2 concentration, CO2 absorption needed to overcome

290

greater mass transfer resistance from liquid phase and gas phase. In short, the higher

291

concentration of CO2 needed a higher amount of the immobilized CA. CO2 absorption

292

faced more resistance, which made the absorption efficiency decline.

293

3.4.3 The effect of gas-flow rate

294

As shown in Fig. 8, the CO2 absorption efficiency first increased and then

295

decreased with the increase in the gas-flow rate. With the low gas-flow rate, the

296

immobilized CA was excessive, so the absorption efficiency increased with the

297

increase of low gas-flow rate

298

and then the absorption efficiency exhibited a downward trend. With the increase in

299

gas-flow rate, the turbulence degree of gas and liquid two phases was improved, so as

300

to realize the full contact of gas and liquid, which made the absorption efficiency

301

increased. But, above 500 mL/min, the increase in the gas-flow rate shortened the

302

residence time of the gas, and therefore, the gas-liquid contact time and the catalytic

303

absorption time with the immobilized CA were reduced [30].

304

3.4.4 The effect of the liquid-to-gas ratio

305

[29]

. However, the curve reached a peak at 500 mL/min,

Fig. 9 shows the CO2 absorption efficiency as a function of the liquid-to-gas ratio.

14


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The absorption efficiency increased from 30.9% to 57.7% with an increase in the

307

liquid-to-gas ratio. When the absorption liquid flow was constant, the change of

308

liquid-to-gas ratio was equivalent to the change of gas-flow rate (shown in Fig. 8).

309

Similarly, the gas-flow rate was constant, and the changing liquid-to-gas ratio was

310

equivalent to the change of liquid flow rate. In this case, the increase in the

311

liquid-to-gas ratio was needed to increase the liquid flow, which could enhance the

312

turbulence of the fluid and the contact of the gas-liquid to make the contact of the

313

gas-liquid more full. Furthermore, increasing the liquid-to-gas ratio could also

314

enhance the mass transfer of the gas-liquid and could be more conducive to CO2

315

absorption. However, with a further increase in the liquid-to-gas ratio, the resistance

316

of the gas-flow rate would increase, due to the existence of water film on the surface

317

of packing, which might influence the mass transfer of the liquid-gas in the tower and

318

hinder CO2 absorption.

319

According to the above results, (a) the absorption efficiency decreased, varying

320

from 63% to 42.7%, with an increase in the concentration of CO2, and the dosage of

321

absorption increased from 75 mg/min to 197 mg/min; (b) the CO2 absorption

322

efficiency first increased and then decreased with an increase in the gas-flow rate; (c)

323

the absorption efficiency increased with an increase in the liquid-to-gas ratio. The

324

optimum conditions were CO2 concentration: 12.73%; gas-flow rate: 500 mL/min;

325

and liquid-to-gas ratio: 32 L/m3.

326

4. Conclusions

327

As a kind of imitation bio-technologies for CO2 capture, CA has been conducted 15


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328

in our research. CA was immobilized on the polyester polymer 80 via the covalent

329

bonding method, and the performance of immobilized CA has been greatly improved.

330

Simultaneously, CO2 absorption efficiency can be promoted effectively with

331

immobilized CA in the vertical reactor. The results were as follows: (1) The optimum

332

conditions of CA immobilized on polyester polyurethane prepolymer 80 were

333

identified when the pH of the Tris-HCl buffer solution, immobilization time, enzyme

334

dose and temperature were 7.5, 24 h, 1 mL enzyme solution/0.1 g carrier and 35 °C,

335

respectively. (2) Compared with free CA, the pH stability, thermal stability,

336

operational stability and storage stability of immobilized CA were significantly

337

improved. (3) The optimum parameters in the CO2 absorption system were as follows:

338

the CO2 concentration, gas-flow rate and liquid-to-gas ratio were 12.73%, 500 mL/s

339

and 32 L/m3, respectively. Under the optimum conditions, the CO2 absorption

340

efficiency reached more than 60% at the low concentration and pressure.

341

From the study, the immobilized CA could enhance the reaction rate of CO2

342

catalytic absorption in the vertical reactor, which establishes a basis for the

343

commercialization of the process of CO2 capture with immobilized CA. Moreover, the

344

experiment was based on a simulated flue gas, but several impurities exist in actual

345

flue gas, such as NOX, SO3 and SO2 et al., which may affect the enzyme catalytic

346

performance. Therefore, the factor of impurities must be considered in subsequent

347

experiments. Meanwhile, the high cost of CA is one of the bottlenecks of industrial

348

application in CO2 capture and storage technology. Extracting CA from animals and

349

plants is an advisable tactic to save the cost of CO2 sequestration in later experiments.

16


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350

Acknowledgments

351

This research is supported by the Doctoral Scientific Fund Project of the

352

Ministry of Education of China (No. 20130092110005), the Open Fund of Key

353

Laboratory of Coal-based CO2 Capture and Geological Storage, Jiangsu Province (No.

354

2016A05), the Natural Science Foundation of China (No. 51176034), the Ordinary

355

University Graduate Student Scientific Research Innovation Projects of Jiangsu

356

province (No. KYLX15-0072) and the Scientific Research Foundation of Graduate

357

School of Southeast University (No. YBJJ1508). The funding is hereby gratefully

358

acknowledged.

359

References

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[1] IEA. http://www.iea.org/publications/freepublications/publication/CO2Emissions

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anhydrase in alginate and its influence on transformation of CO2 to calcite.

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[17] Neto, W., Schürmann, M., Panella, L., Vogel, A., Woodley, J. M. Immobilization

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[19] Bornscheuer., Uwe, T. Immobilizing enzymes: how to create more suitable biocatalysts. Angew. Chem. Int. Ed. 2003, 42 (29), 3336-3337. [20] Sheldon, R. A., Pelt, S. V. Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev. 2013, 42 (15), 6223-35.

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458

Figure captions

459

Fig. 1 Schematic of the CO2 absorption device

460

Fig. 2 The influence of parameters on the enzyme activity. (a) pH; (b) Immobilization

461

time; (c) Temperature; (d) Enzyme dose.

462

Fig. 3 Optimum pH value of immobilized enzyme and free CA

463

Fig. 4 Optimum temperature of immobilized enzyme and free CA

464

Fig. 5 Performance of immobilized CA. (a) pH stability; (b) Thermal stability; (c)

465

Operational stability; (d) Storage stability

466

Fig. 6 Variation characteristic of pH in the absorption process of CO2 with free CA

467

Fig. 7 The effect of the CO2 concentration

468

(Liquid-to-gas ratio: 32 L/m3; gas-flow rate: 500 mL/min; filling height of

469

immobilized CA: 3 cm (0.21 mg))

470

Fig. 8 The effect of gas-flow rate

471

(CO2 concentration: 12.73%; liquid-to-gas ratio: 32 L/m3; filling height of

472


473

Fig. 9 The effect of the liquid-to-gas ratio

474

(CO2 concentration: 12.73%; gas-flow rate: 500 mL/min; filling height of

475


476 477 478 479

22


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Fig. 1 Schematic of the CO2 absorption device

481 482 483 484 485 486 487 488 489 490 491 492 493 494 23


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495

Fig. 2 The influence of parameters on the enzyme activity. (a) pH; (b) Immobilization

496

time; (c) Temperature; (d) Enzyme dose. 70

(a)

60

Enzyme activity recovery (%)


70

50 40 30 20 10 0 7.0

7.5

8.0

8.5

497

40 30 20 10 0

8

12

16

20

24

28

32

Time (h) 70


(c)

60 50 40 30 20 10 0

499

50

9.0

70

498

(b)

60

pH


20

25

30

35

40

(d)

60 50 40 30 20 10 0

45

1

2

Temperature (oC)

3

Enzyme dose (mL)

Fig. 3 Optimum pH value of immobilized enzyme and free CA 110 100

Relative activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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90 80 70 60 50

Free CA Immobilized CA

40 30 7.0

7.5

8.0

8.5

pH

500 501

24


9.0

4

5

Page 25 of 27

502

Fig. 4 Optimum temperature of immobilized enzyme and free CA 110


100 90 80 70 60 50


40 30

20

25

30

35

40

45

Temperature (oC)

503 504

Fig. 5 Performance of immobilized CA. (a) pH stability; (b) Thermal stability; (c)

505

Operational stability; (d) Storage stability 100

100

(a)

(b) 80



90 80 70 60 50


40 30 7.0

7.5

8.0

8.5

60 40

0

9.0


20

20

25

30

35

506

40

45

50

55

60

Temperature (oC)

pH

100

(d)

100

(c)

80

Relative activity (% )

Relative activity (% )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60 40 20

80 60 40

Free CA at 4oC Free CA at room temperature Immobilized CA at 4oC

20

Immobilized CA at room temperature

0

507

1

2

3

4

0

5

0

2

4

Cycle

6

8

Time (d)

508

25


10

12

14

Energy & Fuels

509

Fig. 6 Variation characteristic of pH in the absorption process of CO2 with free CA 7.0 Without free CA in absorbing liquid With immobilized CA in absorbing liquid With free CA in absorbing liquid

pH

6.5

6.0

5.5

0

100

200

300

400

500

Time (d)

510

Fig. 7 The effect of the CO2 concentration

512

(Liquid-to-gas ratio: 32 L/m3; gas-flow rate: 500 mL/min; filling height of

513

immobilized CA: 3 cm (0.21 mg)) 2.0

65

1.6 55

1.4 1.2

50

1.0 45 0.8 40

514

1.8

60

9

10

11

12

13

14

15

CO2 concentration at inlet (%)

515 516 517 518 519 520

26


A b sorp tion qu antity (1 0 2 m g /m in)

511

A b sorp tio n efficien cy (% )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

Page 27 of 27

521

Fig. 8 The effect of the gas-flow rate

522

(CO2 concentration: 12.73%; liquid-to-gas ratio: 32 L/m3; filling height of

523


Absorption efficiency (%)

65

60

55

50

45

200

300

400

500

600

Gas-flow rate (mL/min)

524 525

Fig. 9 The effect of the liquid-to-gas ratio

526

(CO2 concentration: 12.73%; gas-flow rate: 500 mL/min; filling height of

527

immobilized CA: 3 cm (0.21 mg)) 60

Absorption efficiency (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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55 50 45 40 35 30 26

528

28

30

32 3

Liquid-gas ratio (L/m

)

27


34

Investigation on the Immobilization of Carbonic Anhydrase and the

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