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Immobilization of Enterobacter aerogenes on carbon fiber and activated carbon to study hydrogen production enhancement
Accepted Manuscript Title: Immobilization of Enterobacter aerogenes on carbon fiber and activated carbon to study hydrogen production enhancement Authors: Fatemeh Boshagh, Khosrow Rostami, Nasrin Moazami PII: DOI: Reference:
S1369-703X(19)30014-2 https://doi.org/10.1016/j.bej.2019.01.014 BEJ 7136
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
10 October 2018 11 January 2019 12 January 2019
Please cite this article as: Boshagh F, Rostami K, Moazami N, Immobilization of Enterobacter aerogenes on carbon fiber and activated carbon to study hydrogen production enhancement, Biochemical Engineering Journal (2019), https://doi.org/10.1016/j.bej.2019.01.014 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.
Immobilization of Enterobacter aerogenes on carbon fiber and activated carbon to study hydrogen production enhancement Fatemeh Boshagha, Khosrow Rostami, b, Nasrin Moazamib Department of Chemical Technologies, Iranian Research Organization for Science and
Technology (IROST), P.O. Box 3353-5111, Tehran, Iran b
Department of Biotechnology, Iranian Research Organization for Science and Technology
(IROST), P.O. Box 3353-5111, Tehran, Iran
Corresponding author E-mail: Rostami 2002@ yahoo.com , Tel.: +98 21 65276636; fax: +98 21 65276636
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Highlights
Immobilization of E. aerogenes on carbon fiber is addressed for the first time.
Immobilization of E. aerogenes on CF-T resulted in increase of HPR and HY.
Immobilization of E. aerogenes reduces lag phase and generates faster of H2 production
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Abstract
Hydrogen production by Enterobacter aerogenes immobilized on the carbon fiber (CF), surface
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modified carbon fiber, granular and powdered activated carbon in batch mode of operation was investigated. The surface morphology and chemical properties of CFs were characterized by field
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emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). Among the supports employed, immobilization
on treated CF (CF-T) resulted in increase of both hydrogen production rate (HPR) and hydrogen yield (HY). The present study showed that immobilized E. aerogenes on 0.2 mg/mL CF-T resulted in HY of 2.56 mol/mol glucose and HPR of 2.48 L/L.h representing 23%, and 34%, enhancement
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compared to the free E. aerogenes, respectively. Using powdered activated carbon as immobilization support resulted in HY of 2.13 mol/mol glucose, which was higher in comparison to HY of GAC by 1.33 mol/mol glucose.
Keywords: Carbon fiber, Dark fermentation, Enterobacter aerogenes, Immobilization.
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1. Introduction
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The global increasing demand for energy, decreasing of reservoirs of fossil fuels and increasing
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concerns about climate changes, production and usage of renewable energy have become an
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inevitable necessity [1]. Hydrogen as an efficient, renewable, and clean energy carrier is produced from hydrogen-rich feedstock such as water, biomass, or fossil fuel by physicochemical and
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biological methods. The biological hydrogen production methods present the advantages such as
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less energy intensive and performance under mild conditions, which mainly includes photosynthetic and fermentative hydrogen production [2]. Hydrogen via fermentation can be
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produced by methods of dark fermentation through different facultative and obligate anaerobic and
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photo fermentation by using photosynthetic bacteria [3]. The dark fermentation is one of the biological hydrogen production methods that has received increasing attention lately due to its
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higher hydrogen production rate, lower cost, and possibility of applying wastewaters and diverse organic substrates as an inexpensive feedstock [4–6]. The biocatalysts immobilization is an attractive issue in biotechnology processes, which presents several advantages to compare with free biocatalysts, such as more stable, higher resistance against pH and temperature changes and inhibitions, possible to use repeatedly and continuously, and maintenance of higher cell density
during processing [7,8]. There are reports on the dark fermentative hydrogen production employing immobilized cells, which have shown promising results in the improvement of hydrogen production [9–11]. The methods of biocatalysts immobilization include entrapment,
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covalent binding, ionic binding, physical adsorption and the formation of biofilm, granule, and flock [12,13]. The choice of support particle is a key issue of the immobilization process since it depends on various properties including, biocompatibility, hydrophilicity, insolubility, high biomass retention capacity, high chemical, mechanical, and thermal stability, large surface area, low cost, and etc. [14]. The type, particle size, overall shape, and concentration of support can
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affect the immobilization process and subsequently the hydrogen production. The support
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materials shape was usually spheroidal, granular, or cylindrical [15].
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Carbon fiber (CF) contains at least 92 wt % carbon and is attractive for its properties such as
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biocompatibility, light-weight, high stability of chemical, mechanical, and thermal and so on [16].
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The two important precursors of carbon fiber are polyacrylonitrile (PAN) and mesophase pitch
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(MP) [17]. Carbon fiber has potential application in various fields such as catalyst supports [18], microbial fuel cells [19], bioremediation [20], enzyme immobilization [21], and so on. However,
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CF is not widely used as a support for cell immobilization because of its inert surface property, low surface energy and reactivity, smooth surface and hydrophobic nature [22]. In order to enhance
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the properties of the support materials, it is required to modify the support chemical properties by methods such as the plasma treatment, thermal, chemical and electrochemical oxidation, and so on
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[23,24]. The surface oxidation of CFs with nitric acid and nitric and sulfuric acid mixture can increase the number of acidic functional groups on the CFs surface and improve the wettability of the CFs [25], which may effectively improve the cells immobilization.
There are reports available in the literature on types of carbon support such as granular activated carbon (GAC), powdered activated carbon (PAC), cylindrical activated carbon (CAC), carbon nanotube (CNT), and functionalized multi-walled carbon nanotube (MWCNT-COOH)
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have been used in dark fermentative hydrogen production [9,26–41]. Since activated carbon as support material for immobilization of cells provides high specific surface area (1200-1350 m2/g), is favourable for cells attachment and biofilm formation [30]. The methods of biofilm formation and granulation, entrapment, and physical adsorption were used for cells immobilization by carbon supports in dark fermentative hydrogen production. It is known that each method of
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immobilization has certain advantages and disadvantages. For example, entrapment method
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presents disadvantages such as high mass transfer resistance and support breakage by produced
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biogas (when rigid supports were used). Therefore entrapment method may not be a proper choice
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for dark fermentative hydrogen production. Immobilization by attachment to the solid surface may provide low cell loading capacity and the produced biogas can induce cell detachment from the
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support [41]. Wu et al. [26] reported hydrogen production from glucose by seed sludge with two
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immobilization methods of entrapment and granulation. The results showed that HY of granulation
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method was 1.57 mol H2/mol glucose, which was higher than HY of entrapment method by 0.87 mol H2/mol glucose. The three supports of GAC, PAC, and SBA-15 were employed to study dark
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fermentative hydrogen production through biofilm formation by Mohan et al. [35]. The hydrogen yield of SBA-15 was higher in comparison to HY of PAC and GAC. Liu et al. [9] used CNT and
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AC for granulation the anaerobic sludge bacteria in an UASB reactor for hydrogen production. The reactor with CNTs as support presented larger granules, greater flocculation capacity and shorter startup time than that with AC support.
The current study purpose is to investigate the hydrogen production by immobilized Enterobacter aerogenes on carbon fiber of untreated, treated and functionalized by nitric and sulfuric acid mixture, and granular and powdered activated carbon. To the best of our present
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knowledge, PAN-based carbon fiber and its surface modified as immobilization support has not been investigated for the objective of dark fermentative hydrogen production. The hydrogen production rate and hydrogen yield have been compared to various immobilization support in the present study. 2. Materials and Methods
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1.1.Chemicals and Microorganism
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The unidirectional PAN-based T700 carbon fiber with 12000 filament number was
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purchased from Toray Industries Inc. (USA). The diameter, density, and purity of the CFs were 7 μm, 1800 Kg/m3, and 93 %, respectively. Powdered and granular activated carbon was purchased
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from Merck (Germany). About 90 % PAC has particle size < 100 µm and bulk density of
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150 - 440 kg/m3. The size range of granular activated carbon was 2–2.5 mm. All chemicals were of analytical grade and used as received without any further purification and purchased from Merck
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(Germany). Enterobacter aerogenes PTCC 1221 was a gift from the Persian Type Culture
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Collection of Iranian Research Organization for Science and Technology (IROST).
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2.2. Medium and Growth Conditions E. aerogenes was grown in 120 mL serum bottle containing 50 mL of culture medium at
30 °C, pH 6.8, with 2% (v/v) inoculation and without shaking. The complex medium was applied to all experiments. The composition of complex medium consisted of (g/L): glucose as carbon source 20, yeast extract 5, peptone 5, K2HPO4 7, KH2PO4 5.5, (NH4)2SO4 1, MgSO4.7H2O 0.25,
CaCl2.2H2O 0.021, Fe(NH4)2SO4. 6H2O 0.039, NiCl2 0.00002 and 10 mL of trace element solution containing 0.5 g MnC12.4H2O, 0.1 g H3BO3, 0.01 g AlK(SO4)2.H2O, 0.001 g CuCl2.2H2O and 0.5 g Na2EDTA per liter.
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The length of commercially PAN-based carbon fiber (T700) was cut into 20 mm pieces (denoted as CF-0) and immersed in acetone for 3 h, CF was then boiled for 3 h in deionized water, and the deionized water was replaced every 0.5 h to remove the sizing agent coated on the CF surface [42]. The treated CF (denoted as CF-T) was dried at 110 ± 5 ͦ C for 4 h. After treatment, CF was functionalized by using acid oxidation method. In the present study, HNO3 65% and H2SO4
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98% were used. The CF surface was functionalized as follows as 200 mg of CF-T and 40 mL of a
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concentrated acid mixture of HNO3 and H2SO4 at a ratio 1:3 (v/v) was mixed and sonicated in an
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ultrasonic bath for 4.5 h as described by Mubarak et al. [43]. After oxidation, the mixture was
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washed with distilled water to neutral pH and dried at 50± 2 ͦ C for 24 h (designated as CF- NS).
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Before performing experiments, granular and powdered activated carbon were washed several
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times with deionized water and dried at 50± 2 ͦ C for 48 h. 1 mL of seed culture was inoculated into a serum bottle containing 50 mL of culture
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medium at 30°C without shaking. A modified Hungate method in combination with serum bottle
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method was employed to the E. aerogenes culture of anaerobic [44]. After 12 h of incubation, autoclaved immobilization support material (CF-0, CF-T, CF-NS, GAC, and PAC) was immersed
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in the growing culture medium to a final concentration of 0.2, and 0.4 mg/mL and incubated in an incubator shaker at a temperature of 30°C and an agitation rate of 150 rpm for 4 h. This incubation time is required to provide better contact between bacteria and support and enhance cells immobilization on the support. Then, the mixture was centrifuged (5000g, 15 min), and the supernatant was decanted. All experiments were carried out in a 500 mL Erlenmeyer flask
containing 100 mL of fresh complex media, including 10% (v/v) immobilized cells in 0.1 mM potassium phosphate buffer (pH 7). The schematic diagram of the experimental setup is shown in Figure 1. The anaerobic condition was provided by sparging nitrogen gas for 10 min. The reactor
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was placed in a water bath over a magnetic stirrer at a temperature of about 30°C and the rotational speed of 150 rpm. The volume of biogas produced was measured by water displacement method and the batch experiments were continued until biogas production ceased. At the end of each batch, the biomass was centrifuged (5000g, 15 min) and the supernatant was filtered through 0.22 µm mixed cellulose esters (MCE) membranes for the analysis of the volatile fatty acids (VFAs),
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ethanol, and glucose. The control consisted of 100 mL of complex media, including 10% (v/v) of
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inoculum (OD ~2), without support in a 500 mL Erlenmeyer flask.
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The volumetric hydrogen production measured at the ith sampling (VH, i) and the total
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volumetric hydrogen production (VHT) are estimated by Eq. (1) [45]. Where VG,i and VG,i-1 are the
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total biogas volumes at the ith and i-1th sampling, CH,i and CH,i-1 are the fraction of hydrogen gas in
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the reactor headspace measured using gas chromatography at the ith and i-1th sampling, and VH is the total volume of reactor headspace.
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VHT VH ,i 1 CH ,i (VG,i VG,i 1 ) VH (CH ,i CH ,i 1 ) (1)
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2.3. Analysis
The surface physical morphology of all supports before and after immobilization was
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investigated using field emission scanning electron microscopy (FESEM Mira 2, Tescan Co., Brno, Czech Republic). The dehydrated samples by ethanol solutions were sputter-coated with gold before analysis using FESEM. The surface physical morphology of CF support was observed by the transmission electron microscopy (TEM Zeiss - EM10C, 80 kV, Germany). The chemical
composition of CF-T, CF-NS, E.aerogenes and CF-NS-E.aerogenes was investigated using the Fourier transform infrared spectroscopy (FTIR Bruker tensor 27 spectrometer, Germany). The moisture content was measured as the weight (g) of water adsorbed per gram of CF after CF was
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kept at 105°C for 2 h and was cooled in a desiccator until ambient temperature. The CFs (CF-T and CF-NS) surface acidity was measured by mixing 0.04 g of CF with 5 mL of 0.05 M NaOH solution in a closed tube and stirred for 48 h at room temperature. The suspension was discarded and the remaining NaOH was titrated against 0.05 M HCl [46]. The biogas production was measured periodically by the water displacement method at ambient temperature and pressure. The
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hydrogen fraction in the biogas was determined by a gas chromatograph (TG 2550, Iran) equipped
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with a thermal conductivity detector (TCD) and a column packed with 5 A molecular sieves.
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Nitrogen gas was used as carrier gas. The operating temperatures of the column, detector, and
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injector were 65, 100, and 100°C, respectively. Using a 2.5 mL pressure-lock gas-tight syringe (Hamilton, USA) 1 mL of the headspace sample was taken for hydrogen measurement. The pH in
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the medium was measured by a pH meter (526, Germany). The concentration of the volatile fatty
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acids (VFAs) and ethanol (EtOH) was determined using a gas chromatograph (YL 6500, Korea)
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equipped with a flame ionization detector (FID) and a TRB-G43 capillary column (30m× 0.53mm ×3µm). The operating temperatures of the injector and the detector were 250°C. The initial
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temperature of the column was 70 °C for 3 min and at a rate of 20° C/ min was increased to 150°C for 3 min. Nitrogen was applied as the carrier gas at a flow rate of 10 mL/min. The concentration
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of glucose was determined by the DNS colorimetric method at a λmax of 540 nm [47]. 2.4. Kinetic modeling Modified Gompertz model (Eq. (2)) [1] was employed to predict the hydrogen production in the batch experiments.
R e H H max exp exp max t 1 H max
(2)
Where H (mL), Hmax (mL), and Rmax (mL/h) are the cumulative hydrogen production, the hydrogen
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production potential, and the maximum hydrogen production rate, respectively. λ (h) and t (h) are the lag phase and incubation time and e is 2.71828. The kinetic parameters (Hmax, Rmax, λ, and R2) were evaluated using the software OriginPro 9.1. 2. Results and Discussion
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The chemical properties of CF-T and CF-NS samples were checked by Fourier transform
represents the OH stretching vibrations and peaks appearing at 1634 cm-1 and 1380 cm-1 indicated
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infrared (FTIR) spectrophotometer as shown in Fig. 2 (A), (B), respectively. The peak at 3423 cm-
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C=O stretching, and C–O stretching in carboxylic groups, and carboxylate moieties, respectively. The peaks of CF-NS are stronger than CF-T, which indicates that the oxidation treatment gave rise
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to an increase of oxygen-containing groups. The results of the current study are similar to the
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finding of Bao et al. [48] and Wang et al. [49]. FTIR spectrum for E.aerogenes is shown in Fig. 2 (C), which the peak at 3295 cm−1 indicates the N-H and O-H stretching vibration: polysaccharides,
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proteins. Peaks at 2959 cm−1 and 2926 cm−1 illustrate the CH3 asymmetric stretch: mainly lipids
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and the CH2 asymmetric stretch: mainly lipids, with little contribution from proteins, carbohydrates, and nucleic acids, respectively. The peak at 2854 cm−1 shows the CH3 symmetric
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stretch: largely proteins, with little contribution from lipids, carbohydrates, and nucleic acids. The peak at 2851 cm−1 displays the CH2 symmetric stretch: mainly lipids, with little contribution from proteins, carbohydrates, and nucleic acids. Peaks at 1759 cm−1 and 1384 cm−1 represent the ester C=O stretch: lipid, triglycerides and C-O symmetric stretch: amino acid side chains, fatty acids, respectively. Peaks at 1659 cm−1 and 1539 cm−1 demonstrate the amide (protein C=O stretching):
α helices, and the amide (protein N-H bend, C-N stretch): α helices. The peak at 1452 cm−1 shows the CH2 bending: lipids and the peak at 1237cm−1 indicates the PO-2 asymmetric stretching: mainly nucleic acids with little contribution from phospholipids. Peaks at 1150 cm−1 exhibit the CO-O-C
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asymmetric stretching: glycogen and nucleic acids, the peak at 1077 cm−1 depicts PO-2 asymmetric stretching: mainly nucleic acids with a little contribution from phospholipids and the peak at 948 cm−1 displays the C-N+-C stretch: nucleic acids. The present results have good agreement with the reports by Davis and Mauer [50]. FTIR spectrum for CF-NS– E.aerogenes is illustrated in Fig. 2 (D). Peak at 3289 cm−1 exhibits the combination of stretching vibration of N - H and O - H. The
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peaks at 1648 cm−1, 1532 cm−1, and 2358 cm−1 illustrate C = O stretching mode of amid group, N
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- H mode of amid group and C ≡ N mode of nitriles group, which indicates that E.aerogenes was
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successfully immobilized on the CF-NS by covalent binding.
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The surface morphology of CF supports using TEM and FESEM at different magnification
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scales are observed in Figs. 3 and 4, respectively. The TEM analysis shows that bacteria attach
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apparently on the CFs surfaces. As shown in Fig. 3-A4, the increment in the thickness of CFs after immobilization illustrate the presence of E. aerogenes on the surface of CFs. FESEM of CF
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samples in different magnification scales before and after cells immobilization is shown in Fig. 4. Figs. 4 A1, and A2 depict CF-0 before cells immobilization and Figs. 4 A3, and A4 indicate CF-0
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after cells immobilization. Figs. 4 B1 demonstrate CF-T before cells immobilization and Figs. 4 B2, B3, and B4 exhibit CF-T after cells immobilization. Figs. 4 C1 illustrate CF-NS before cells
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immobilization and Figs. 4, C2, C3, and C4 display CF-NS after cells immobilization. It can be concluded that the treatment of CF improved support properties and more cells could be immobilized on the surface of the CF-T. As shown in Fig. 4 immobilized cells loading amount is in the form of CF-T > CF-NS > CF-0. The surface morphology of PAC and GAC samples before
and after cells immobilization using FESEM at 1 µm scale is observed in Fig. 5. Figs. 5 A1 and A2 represent GAC before cells immobilization and Figs. 5 A3 and A4 after cells immobilization. Figs. 5 B1 and B2 illustrate PAC before cells immobilization and Figs. 5 B, and B4 show PAC
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after cells immobilization. For biofilm formation, a microorganism should encounter and attach a suitable surface and grow on the surface to produce a stable biofilm. The physical and chemical characteristics of the surface and the surrounding medium can affect biofilm formation [51]. Attachment of cells and development of biofilm on supports is related to the surface properties such as wettability,
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morphology and roughness, surface energy, electrical properties, and chemical composition of the
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supports [49]. The content of acidic functional groups and the moisture content value in CF-NS
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was higher than the corresponding values in CF-T. Total acidity and the moisture content values
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were %7 and 6.73 mmol/g for CF-NS and %2.6 and 3.49 mmol/g for CF-T, respectively. The
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present result shows that the wettability of CFs was increased with the increasing of hydrophilic
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functional groups on the CFs surface.
The mean mass of attached biomass on the support material was evaluated as the specific
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dry weight (g) of immobilized microorganisms on 0.01 g support at the end of fermentation, which
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was applied as a parameter to show the affinity between microorganism and support for biofilm formation or physical adsorption. The immobilized cells on CF-0, CF-T, and CF-NS, were 18.5,
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32.6, and 28.9 g/g, respectively. While the immobilized cells on PAC and GAC were 23.2 g/g and 13.9 g/g, respectively. Therefore, CF-T support was achieved high cell loading capacity. It is necessary for the support to immobilize high amount of biomass and provide conditions for efficient hydrogen production [52].
The HY, HPR, total volatile fatty acids (TVFAs) and ethanol yield, final pH, and glucose degradation efficiency (%) in the batch mode of operation for free and immobilized cells have been exhibited in Tables 1 and 2. As shown in Table 1 HY and HPR of free cells were 2.09 mol/mol
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glucose and 1.85 L/h. L, respectively. Hydrogen yield and hydrogen production rate of immobilized E. aerogenes on 0.2 mg/mL CF-0, CF-T, and CF-NS were 1.83, 2.56, and 2.11 mol/mol glucose, and 2.22, 2.48 and 2.10 L/h.L, respectively. The carbon fiber surface was functionalized to improve hydrophilicity and biocompatibility, however, a surface of very hydrophilic can also hinder immobilization of microorganism [48]. It seems microorganisms
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prefer to adhere to support surfaces with moderate hydrophilicity. Immobilization of cells on 0.2
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mg/mL of PAC and GAC resulted in HY of 2.13 and 1.33 mol/mol glucose and HPR of 2.51 and
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2.11 L/h.L, respectively. Comparison of various activated carbon as support materials (PAC and
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GAC) showed that PAC has higher HY and HPR. Powdered activated carbon has a relatively smaller particle size when compared with granular activated carbons and consequently, presents a
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larger surface to volume ratio. During the investigation of effect of support concentration, results
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showed that at lower concentration of all supports immobilized E. aerogenes was more effective,
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which may describe that mass transfer resistance was higher in high concentration of support material. Excessive amount of supports material may reduce contact between bacteria with
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substrate and nutrients [53]. Immobilization of E. aerogenes on GAC, PAC, and CFs resulted in the shorter lag phase compared to the control. The glucose degradation efficiency of immobilized
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cells of E. aerogenes on supports was lower than free cells. The glucose degradation efficiency when using CFs was 71-77%, which was lower compared to the control (95%). In the cases of PAC and GAC, the glucose degradation efficiency was 71-82%.
The main soluble metabolites measured during dark fermentation in the present study are butyric acid (HBu), propionic acid (HPr), acetic acid (HAc) and ethanol (EtOH), while similar results are published by Mohanraj et al. [54] and Lin et al. [55]. In free cells system, butyric acid
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is the primary VFAs product, followed by propionic acid and acetic acid. As shown in Table 2, HBu, HPr, and HAc for free E. aerogenes are 2907, 884, and 570 mg/ L, respectively. Using 0.2 mg/mL CF-T as support, HBu, HPr, and HAc are 1880, 374, and 336 mg/L, respectively. During dark fermentation of carbohydrates for hydrogen production, the pathways indicated that production of acetic and butyric acids releases hydrogen into the medium according to Eqs. (3)
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and (4). The overall equation for the production of propionic acid from glucose (Eq. (5)), showing
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that it involves consumption of hydrogen [56]. Ethanol as by-product of a metabolic pathway
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produces metabolites, which compete with hydrogen producing. When ethanol is produced, at least
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2mol of NAD+ are released. NAD+ can later on be recycled back to NADH in a reaction competing
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metabolites is unfavourable.
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with hydrogen production [10]. Therefore, presence of ethanol and propionic acid in end
(3)
C6H12O6 + 2H2O 2CH3COOH + 4H2 + 2CO2
(4)
C6H12O6 + 2H2 2CH3CH2COOH + 2H2O
(5)
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C6H12O6 CH3CH2CH2COOH + 2H2 + 2CO2
The concentration of 0.2 mg/mL CF-T was selected for the reusability study of
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immobilized cells of E. aerogenes in repeated batch experiments. As demonstrated in Table 3 after the sixth batch, HY decreased to 0.91 mol/mol glucose and HPR increased to 2.97 L/h.L. Table 3 shows a 1.34–1.61-fold enhancement in HPR from 1st to 6th batch when compared to the control. However, HY was decreased from 2.56 in 1st batch to 0.91 in 6th batch.
The cumulative hydrogen data are fitted to modified Gompertz model and the kinetic parameters are presented in Table 3. As illustrated in Table 4 and Fig. 6 the correlation coefficients (R2) of all the regressions are over 0.97, which illustrate that the modified Gompertz model fitted
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the data very well and estimated the progress of volumetric hydrogen production in the current study successfully. Table 4 reveals that the maximum hydrogen production potential (Hmax) for free E. aerogenes and immobilized E. aerogenes on 0.2 mg/mL CF-T by modified Gompertz model is 178 mL and 191 mL, respectively. Further, the aforesaid values are in good agreement with the experimental values of the present study.
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In the current study, treated and functionalized carbon fiber were used for the first time
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successfully for immobilization of E. aerogenes in dark fermentative hydrogen production.
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Comparison of three CFs (CF-0, CF-T, and CF-NS) as support for immobilization illustrated that
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CF-T provides higher biomass immobilization capacity, and consequently higher HY and HPR
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than CF-0 and CF-NS. The treated carbon fiber surface provides moderate hydrophilicity that is
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effective for cells immobilization. Immobilization of E. aerogenes on the surface of treated carbon fiber and functionalized carbon fiber is carried out by biofilm formation and covalent binding,
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respectively. The results show that HY of biofilm formation method is higher than HY of covalent binding method. The covalent binding provides durable attachment compared to biofilm
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formation, however, the strong binding may also inhibits cells movement and resulted in reduce their activity [7]. Comparison between activated carbon supports (PAC and GAC), showed that
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immobilization on PAC results in higher HY and HPR. Powdered activated carbon presents a smaller particle size (100 µm) when compared to GAC (2-2.5 mm) and provides a larger surface area and consequently, shows higher biomass immobilization capacity. Mohan et al. [35] employed GAC and PAC as supports to enhance dark fermentative hydrogen production and
indicated HY of GAC was higher in comparison to HY of PAC .The most relevant studies are shown in Table 5 and is concluded that the application of carbon supports is a promising method to improve hydrogen production from various substrates.
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3. Conclusions The current study has attempted to investigate biohydrogen production by immobilized E. aerogenes on the carbon fiber, surface modified carbon fiber, and granular and powdered activated carbon with an objective of enhancing biohydrogen production. Immobilization of E. aerogenes on CF, PAC and GAC has almost economic advantages such as reducing lag phase, generating
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faster of hydrogen production over a time period and supports are stable in the medium. Hydrogen
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yield of immobilized E. aerogenes on 0.2 mg/mL CF-0, CF-T, and CF-NS was 1.83, 2.56, and
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2.11 mol/mol glucose, respectively. While PAC and GAC resulted in HY of 2.13 and 1.33 mol/mol
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aerogenes to produce biohydrogen.
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glucose. The results indicate that CF may be used as a promising support for immobilization of E.
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Acknowledgements
The authors would like to express their gratitude to Iran National Science Foundation
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(Grant no. 93012717) for the financial support of the present study.
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Schematic
diagram
A
CC
EP
TE
D
Figure1
M
A
N
U
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Figures and Tables
of
the
experimental
set
up
SC RI PT U N A M D TE EP
CC
Figure2. FTIR spectra of CF-T (A), CF-NS (B), Enterobacter aerogenes (C),
A
CF-NS- Enterobacter aerogenes (D)
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Figure 3.TEM of CF-T -E. aerogenes conjugate in different magnification scales: 200 nm (A), 1
A
CC
EP
TE
µm (B) and (C), and 500 nm (D).
SC RI PT U N A M D TE
EP
Figure 4. FESEM image of CFs samples in different magnification scales before and after cells immobilization. A1 (5 µm), and A2 (100 µm) indicated CF-0 before and A3 (100 µm) and A4 (10
CC
µm) indicated CF-0 after cells immobilization. B1 (5 µm) indicated CF-T before and B2 (200 µm), B3 (100 µm), and B4 (10 µm) indicated CF-T after cells immobilization. C1 (5 µm) indicated CF-
A
NS before and C2 (200 µm), C3 (100 µm), and C4 (10 µm) indicated CF-NS after cells immobilization.
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M
Figure 5. FESEM image of GAC and PAC in 1 µm scale. A1, A2, indicated GAC before and A3
A
CC
EP
TE
after cells immobilization.
D
and A4 after cells immobilization. B1 and B2 indicated PAC before and B3 and B4 indicated PAC
SC RI PT U N A M D TE
A
CC
EP
Fig. 6 .Kinetic analysis of batch experiments by modified Gompertz model
Table 1- Performance in the batch mode of operation Support
Type
(mg/mL)
glucose)
(L/h.L)
Control
0
2.09±0.46
PAC
0.2
CF- T
Final pH
1.85± 0.41
95
5.52±0.02
2.13±0.22
2.51±0.34
82
5.95±0.04
0.4
2.04±0.31
2.25±0.23
79
5.83±0.03
0.2
1.33±0.37
2.11±0.29
72
5.58±0.03
0.4
1.27±0.38
2.08±0.38
71
5.77±0.02
0.2
1.83±0.41
2.22±0.47
75
5.60±0.03
0.4
1.81±0.38
2.03±0.42
74
5.62±0.01
2.56±0.34
2.48±0.36
77
5.55±0.04
2.29±0.39
2.27±0.37
76
5.61±0.02
2.11±0.49
2.10±0.31
74
5.58±0.02
1.85±0.42
1.76±0.29
71
5.41±0.02
0.2
EP
0.2
TE
0.4 CF- NS
A
CC
0.4
U
N
A
M
CF-0
EG (%)
D
GAC
concentration HY (mol /mol HPR
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Support
Table 2- Produced end metabolites in the batch mode of operation Support concentration (mg/mL) End-metabolites (mg/L)
HAc
HPr
HBu
0
1331±19
570±13
884±26
2907±32
PAC
0.2
1706±27
237±8
395±20
3116±24
0.4
1897±14
541±11
412±9
2271±27
0.2
2036±21
396±14
305±39
1378±15
0.4
2252±35
452±17
660±16
2085±41
0.2
3659±28
421±22
647±33
3061±14
3090±37
499±18
440±9
2117±25
1025±26
336±25
374±17
1880±31
1627±18
532±23
475±32
2628±39
1033±23
679±29
253±13
1132±32
941±11
726±15
260±8
716±21
CF-0
M
GAC
N
Control
U
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EtOH
A
Support Type
0.4
0.2
EP
CF-NS
TE
0.4
D
0.2
CF-T
A
CC
0.4
Table 3- Reusability of immobilized E. aerogenes on 0.2 mg/mL CF-T HY
batch NO.
(mol
HPR
Yields of end metabolites (mg/L)
EG
/mol (L/h.L) HAc
HPr
HBu
1
2.56±0.34
2.48±0.36
1025±26
336±25
374±17
1880±31
77
5.55±0.04
2
2.14±0.31
2.53±0.22
2342.62
627.56
421.42
2155.45
76
5.12±0.05
3
1.99±0.29
2.57±0.21
1184.53
827.84
251.55
2574.02
72
5.08±0.03
4
1.56±0.26
2.64±0.39
1118.79
440.63
U
EtOH
1396.32
75
5.05±0.07
5
1.27±0.33
2.89±0.41
1454.91
322.68
320.15
2660.81
79
4.89±0.08
6
0.91±0.22
2.97±0.44
975.81
A
glucose)
Final pH
(%)
SC RI PT
Fermentation
881.25
315.25
3063.99
78
5.43±0.03
N
M D TE EP CC A
267.88
Table 4- Kinetic parameters of hydrogen production for free and immobilized E. aerogenes Modified Gompertz model Rmax (mL/h)
λ (h)
R2
Control
178
104
3.2
0.9984
0.2 mg/mL CF-0
154
72
1
0.9883
0.2 mg/mL CF-T
191
82
0.9
0.9855
0.2 mg/mL CF-NS
185
82
0.9
0.9786
0.2 mg/mL PAC
188
88
0.2 mg/mL GAC
150
N
U
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Hmax (mL)
M
Fermentation batch
0.9915
1.1
0.9893
A
1
A
CC
EP
TE
D
65
Table 5- Comparative studies on types of carbon support employed in dark fermentative hydrogen production Substrate
Support
Immobilization
type
method Physical adsorption 1.90-3.76 mol/mol sucrose
Seed sludge
Sucrose
AC
E.aerogenes
Glucose
MWCNT- Covalent binding COOH
Glucose
CF-NS
Covalent binding
Glucose
PAC
N
Biofilm
A
CF-T
Biofilm
Glucose
1.85-2.11 mol/mol glucose
Present
2.29-2.56 mol/mol glucose
TE
GAC
study Present study
2.04-2.13 mol/mol glucose
Present study
Biofilm
1.27-1.33 mol/mol glucose
Present study
AC
Granulation
1.42 mol/mol glucose
[9]
Glucose
CNT
Granulation
2.45 mol/mol glucose
[9]
Seed sludge
Glucose
AC
Granulation
1.57 mol/mol glucose
[26]
Seed sludge
Glucose
AC
Entrapment
0.87 mol/mol glucose
[26]
Seed sludge
Sucrose
AC
Entrapment
2.62 mol/mol sucrose
[33]
CC
Anaerobic sludge
Glucose
EP
E.aerogenes
[32] [27]
D
E.aerogenes
Glucose
M
E.aerogenes
Ref.
2.14 -2.2 mol/mol glucose
U
E.aerogenes
H2 yield
SC RI PT
Microorganism
A
Anaerobic sludge
Seed sludge
Sucrose
AC
Entrapment
1.82-4.98 mol/mol sucrose
Seed sludge
Chemical
GAC
Biofilm
1.28-2.38 mol/kg
wastewater
day
Chemical
PAC
Biofilm
1.22-1.57 mol/kg day
Biofilm
Seed sludge
Sucrose
CAC
Biofilm
Seed sludge
Glucose
GAC
Biofilm
Seed sludge
Glucose & xylose
GAC
Biofilm
Seed sludge
Sucrose
CAC
Biofilm
Sucrose
CAC
Seed sludge
Glucose
Seed sludge
Molasses
+
E.
A
homaechei 83
[36]
-
[37]
0.94-1.19 mol/mol glucose
[38]
0.31-1.01 mol/mol sugar
[39]
1.01-2.45 mol/mol sucrose
[40]
Biofilm
1.01-2.45 mol/mol sucrose
[28]
Biofilm
0.5-1.7 mol/mol glucose
[29]
GAC
Biofilm
3.47 mol/mol sucrose
[30]
AC
Biofilm
0.3-1.2 mol/mol glucose
[31]
TE
GAC
EP
2A
CC
HG6
Glucose
1.2-3.9 mol/mol sucrose
U
CAC
N
Sucrose
M
A
Seed sludge
E.cancerogeous
CODR- [35]
D
wastewater
Seed sludge
CODR- [35]
SC RI PT
Seed sludge
[34]
S1369-703X(19)30014-2 https://doi.org/10.1016/j.bej.2019.01.014 BEJ 7136
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
10 October 2018 11 January 2019 12 January 2019
Please cite this article as: Boshagh F, Rostami K, Moazami N, Immobilization of Enterobacter aerogenes on carbon fiber and activated carbon to study hydrogen production enhancement, Biochemical Engineering Journal (2019), https://doi.org/10.1016/j.bej.2019.01.014 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.
Immobilization of Enterobacter aerogenes on carbon fiber and activated carbon to study hydrogen production enhancement Fatemeh Boshagha, Khosrow Rostami, b, Nasrin Moazamib Department of Chemical Technologies, Iranian Research Organization for Science and
Technology (IROST), P.O. Box 3353-5111, Tehran, Iran b
Department of Biotechnology, Iranian Research Organization for Science and Technology
(IROST), P.O. Box 3353-5111, Tehran, Iran
Corresponding author E-mail: Rostami 2002@ yahoo.com , Tel.: +98 21 65276636; fax: +98 21 65276636
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a
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Highlights
Immobilization of E. aerogenes on carbon fiber is addressed for the first time.
Immobilization of E. aerogenes on CF-T resulted in increase of HPR and HY.
Immobilization of E. aerogenes reduces lag phase and generates faster of H2 production
TE
D
M
A
EP
Abstract
Hydrogen production by Enterobacter aerogenes immobilized on the carbon fiber (CF), surface
CC
modified carbon fiber, granular and powdered activated carbon in batch mode of operation was investigated. The surface morphology and chemical properties of CFs were characterized by field
A
emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). Among the supports employed, immobilization
on treated CF (CF-T) resulted in increase of both hydrogen production rate (HPR) and hydrogen yield (HY). The present study showed that immobilized E. aerogenes on 0.2 mg/mL CF-T resulted in HY of 2.56 mol/mol glucose and HPR of 2.48 L/L.h representing 23%, and 34%, enhancement
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compared to the free E. aerogenes, respectively. Using powdered activated carbon as immobilization support resulted in HY of 2.13 mol/mol glucose, which was higher in comparison to HY of GAC by 1.33 mol/mol glucose.
Keywords: Carbon fiber, Dark fermentation, Enterobacter aerogenes, Immobilization.
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1. Introduction
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The global increasing demand for energy, decreasing of reservoirs of fossil fuels and increasing
A
concerns about climate changes, production and usage of renewable energy have become an
M
inevitable necessity [1]. Hydrogen as an efficient, renewable, and clean energy carrier is produced from hydrogen-rich feedstock such as water, biomass, or fossil fuel by physicochemical and
D
biological methods. The biological hydrogen production methods present the advantages such as
TE
less energy intensive and performance under mild conditions, which mainly includes photosynthetic and fermentative hydrogen production [2]. Hydrogen via fermentation can be
EP
produced by methods of dark fermentation through different facultative and obligate anaerobic and
CC
photo fermentation by using photosynthetic bacteria [3]. The dark fermentation is one of the biological hydrogen production methods that has received increasing attention lately due to its
A
higher hydrogen production rate, lower cost, and possibility of applying wastewaters and diverse organic substrates as an inexpensive feedstock [4–6]. The biocatalysts immobilization is an attractive issue in biotechnology processes, which presents several advantages to compare with free biocatalysts, such as more stable, higher resistance against pH and temperature changes and inhibitions, possible to use repeatedly and continuously, and maintenance of higher cell density
during processing [7,8]. There are reports on the dark fermentative hydrogen production employing immobilized cells, which have shown promising results in the improvement of hydrogen production [9–11]. The methods of biocatalysts immobilization include entrapment,
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covalent binding, ionic binding, physical adsorption and the formation of biofilm, granule, and flock [12,13]. The choice of support particle is a key issue of the immobilization process since it depends on various properties including, biocompatibility, hydrophilicity, insolubility, high biomass retention capacity, high chemical, mechanical, and thermal stability, large surface area, low cost, and etc. [14]. The type, particle size, overall shape, and concentration of support can
U
affect the immobilization process and subsequently the hydrogen production. The support
N
materials shape was usually spheroidal, granular, or cylindrical [15].
A
Carbon fiber (CF) contains at least 92 wt % carbon and is attractive for its properties such as
M
biocompatibility, light-weight, high stability of chemical, mechanical, and thermal and so on [16].
D
The two important precursors of carbon fiber are polyacrylonitrile (PAN) and mesophase pitch
TE
(MP) [17]. Carbon fiber has potential application in various fields such as catalyst supports [18], microbial fuel cells [19], bioremediation [20], enzyme immobilization [21], and so on. However,
EP
CF is not widely used as a support for cell immobilization because of its inert surface property, low surface energy and reactivity, smooth surface and hydrophobic nature [22]. In order to enhance
CC
the properties of the support materials, it is required to modify the support chemical properties by methods such as the plasma treatment, thermal, chemical and electrochemical oxidation, and so on
A
[23,24]. The surface oxidation of CFs with nitric acid and nitric and sulfuric acid mixture can increase the number of acidic functional groups on the CFs surface and improve the wettability of the CFs [25], which may effectively improve the cells immobilization.
There are reports available in the literature on types of carbon support such as granular activated carbon (GAC), powdered activated carbon (PAC), cylindrical activated carbon (CAC), carbon nanotube (CNT), and functionalized multi-walled carbon nanotube (MWCNT-COOH)
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have been used in dark fermentative hydrogen production [9,26–41]. Since activated carbon as support material for immobilization of cells provides high specific surface area (1200-1350 m2/g), is favourable for cells attachment and biofilm formation [30]. The methods of biofilm formation and granulation, entrapment, and physical adsorption were used for cells immobilization by carbon supports in dark fermentative hydrogen production. It is known that each method of
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immobilization has certain advantages and disadvantages. For example, entrapment method
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presents disadvantages such as high mass transfer resistance and support breakage by produced
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biogas (when rigid supports were used). Therefore entrapment method may not be a proper choice
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for dark fermentative hydrogen production. Immobilization by attachment to the solid surface may provide low cell loading capacity and the produced biogas can induce cell detachment from the
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support [41]. Wu et al. [26] reported hydrogen production from glucose by seed sludge with two
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immobilization methods of entrapment and granulation. The results showed that HY of granulation
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method was 1.57 mol H2/mol glucose, which was higher than HY of entrapment method by 0.87 mol H2/mol glucose. The three supports of GAC, PAC, and SBA-15 were employed to study dark
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fermentative hydrogen production through biofilm formation by Mohan et al. [35]. The hydrogen yield of SBA-15 was higher in comparison to HY of PAC and GAC. Liu et al. [9] used CNT and
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AC for granulation the anaerobic sludge bacteria in an UASB reactor for hydrogen production. The reactor with CNTs as support presented larger granules, greater flocculation capacity and shorter startup time than that with AC support.
The current study purpose is to investigate the hydrogen production by immobilized Enterobacter aerogenes on carbon fiber of untreated, treated and functionalized by nitric and sulfuric acid mixture, and granular and powdered activated carbon. To the best of our present
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knowledge, PAN-based carbon fiber and its surface modified as immobilization support has not been investigated for the objective of dark fermentative hydrogen production. The hydrogen production rate and hydrogen yield have been compared to various immobilization support in the present study. 2. Materials and Methods
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1.1.Chemicals and Microorganism
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The unidirectional PAN-based T700 carbon fiber with 12000 filament number was
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purchased from Toray Industries Inc. (USA). The diameter, density, and purity of the CFs were 7 μm, 1800 Kg/m3, and 93 %, respectively. Powdered and granular activated carbon was purchased
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from Merck (Germany). About 90 % PAC has particle size < 100 µm and bulk density of
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150 - 440 kg/m3. The size range of granular activated carbon was 2–2.5 mm. All chemicals were of analytical grade and used as received without any further purification and purchased from Merck
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(Germany). Enterobacter aerogenes PTCC 1221 was a gift from the Persian Type Culture
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Collection of Iranian Research Organization for Science and Technology (IROST).
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2.2. Medium and Growth Conditions E. aerogenes was grown in 120 mL serum bottle containing 50 mL of culture medium at
30 °C, pH 6.8, with 2% (v/v) inoculation and without shaking. The complex medium was applied to all experiments. The composition of complex medium consisted of (g/L): glucose as carbon source 20, yeast extract 5, peptone 5, K2HPO4 7, KH2PO4 5.5, (NH4)2SO4 1, MgSO4.7H2O 0.25,
CaCl2.2H2O 0.021, Fe(NH4)2SO4. 6H2O 0.039, NiCl2 0.00002 and 10 mL of trace element solution containing 0.5 g MnC12.4H2O, 0.1 g H3BO3, 0.01 g AlK(SO4)2.H2O, 0.001 g CuCl2.2H2O and 0.5 g Na2EDTA per liter.
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The length of commercially PAN-based carbon fiber (T700) was cut into 20 mm pieces (denoted as CF-0) and immersed in acetone for 3 h, CF was then boiled for 3 h in deionized water, and the deionized water was replaced every 0.5 h to remove the sizing agent coated on the CF surface [42]. The treated CF (denoted as CF-T) was dried at 110 ± 5 ͦ C for 4 h. After treatment, CF was functionalized by using acid oxidation method. In the present study, HNO3 65% and H2SO4
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98% were used. The CF surface was functionalized as follows as 200 mg of CF-T and 40 mL of a
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concentrated acid mixture of HNO3 and H2SO4 at a ratio 1:3 (v/v) was mixed and sonicated in an
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ultrasonic bath for 4.5 h as described by Mubarak et al. [43]. After oxidation, the mixture was
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washed with distilled water to neutral pH and dried at 50± 2 ͦ C for 24 h (designated as CF- NS).
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Before performing experiments, granular and powdered activated carbon were washed several
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times with deionized water and dried at 50± 2 ͦ C for 48 h. 1 mL of seed culture was inoculated into a serum bottle containing 50 mL of culture
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medium at 30°C without shaking. A modified Hungate method in combination with serum bottle
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method was employed to the E. aerogenes culture of anaerobic [44]. After 12 h of incubation, autoclaved immobilization support material (CF-0, CF-T, CF-NS, GAC, and PAC) was immersed
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in the growing culture medium to a final concentration of 0.2, and 0.4 mg/mL and incubated in an incubator shaker at a temperature of 30°C and an agitation rate of 150 rpm for 4 h. This incubation time is required to provide better contact between bacteria and support and enhance cells immobilization on the support. Then, the mixture was centrifuged (5000g, 15 min), and the supernatant was decanted. All experiments were carried out in a 500 mL Erlenmeyer flask
containing 100 mL of fresh complex media, including 10% (v/v) immobilized cells in 0.1 mM potassium phosphate buffer (pH 7). The schematic diagram of the experimental setup is shown in Figure 1. The anaerobic condition was provided by sparging nitrogen gas for 10 min. The reactor
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was placed in a water bath over a magnetic stirrer at a temperature of about 30°C and the rotational speed of 150 rpm. The volume of biogas produced was measured by water displacement method and the batch experiments were continued until biogas production ceased. At the end of each batch, the biomass was centrifuged (5000g, 15 min) and the supernatant was filtered through 0.22 µm mixed cellulose esters (MCE) membranes for the analysis of the volatile fatty acids (VFAs),
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ethanol, and glucose. The control consisted of 100 mL of complex media, including 10% (v/v) of
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inoculum (OD ~2), without support in a 500 mL Erlenmeyer flask.
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The volumetric hydrogen production measured at the ith sampling (VH, i) and the total
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volumetric hydrogen production (VHT) are estimated by Eq. (1) [45]. Where VG,i and VG,i-1 are the
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total biogas volumes at the ith and i-1th sampling, CH,i and CH,i-1 are the fraction of hydrogen gas in
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the reactor headspace measured using gas chromatography at the ith and i-1th sampling, and VH is the total volume of reactor headspace.
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VHT VH ,i 1 CH ,i (VG,i VG,i 1 ) VH (CH ,i CH ,i 1 ) (1)
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2.3. Analysis
The surface physical morphology of all supports before and after immobilization was
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investigated using field emission scanning electron microscopy (FESEM Mira 2, Tescan Co., Brno, Czech Republic). The dehydrated samples by ethanol solutions were sputter-coated with gold before analysis using FESEM. The surface physical morphology of CF support was observed by the transmission electron microscopy (TEM Zeiss - EM10C, 80 kV, Germany). The chemical
composition of CF-T, CF-NS, E.aerogenes and CF-NS-E.aerogenes was investigated using the Fourier transform infrared spectroscopy (FTIR Bruker tensor 27 spectrometer, Germany). The moisture content was measured as the weight (g) of water adsorbed per gram of CF after CF was
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kept at 105°C for 2 h and was cooled in a desiccator until ambient temperature. The CFs (CF-T and CF-NS) surface acidity was measured by mixing 0.04 g of CF with 5 mL of 0.05 M NaOH solution in a closed tube and stirred for 48 h at room temperature. The suspension was discarded and the remaining NaOH was titrated against 0.05 M HCl [46]. The biogas production was measured periodically by the water displacement method at ambient temperature and pressure. The
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hydrogen fraction in the biogas was determined by a gas chromatograph (TG 2550, Iran) equipped
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with a thermal conductivity detector (TCD) and a column packed with 5 A molecular sieves.
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Nitrogen gas was used as carrier gas. The operating temperatures of the column, detector, and
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injector were 65, 100, and 100°C, respectively. Using a 2.5 mL pressure-lock gas-tight syringe (Hamilton, USA) 1 mL of the headspace sample was taken for hydrogen measurement. The pH in
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the medium was measured by a pH meter (526, Germany). The concentration of the volatile fatty
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acids (VFAs) and ethanol (EtOH) was determined using a gas chromatograph (YL 6500, Korea)
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equipped with a flame ionization detector (FID) and a TRB-G43 capillary column (30m× 0.53mm ×3µm). The operating temperatures of the injector and the detector were 250°C. The initial
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temperature of the column was 70 °C for 3 min and at a rate of 20° C/ min was increased to 150°C for 3 min. Nitrogen was applied as the carrier gas at a flow rate of 10 mL/min. The concentration
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of glucose was determined by the DNS colorimetric method at a λmax of 540 nm [47]. 2.4. Kinetic modeling Modified Gompertz model (Eq. (2)) [1] was employed to predict the hydrogen production in the batch experiments.
R e H H max exp exp max t 1 H max
(2)
Where H (mL), Hmax (mL), and Rmax (mL/h) are the cumulative hydrogen production, the hydrogen
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production potential, and the maximum hydrogen production rate, respectively. λ (h) and t (h) are the lag phase and incubation time and e is 2.71828. The kinetic parameters (Hmax, Rmax, λ, and R2) were evaluated using the software OriginPro 9.1. 2. Results and Discussion
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The chemical properties of CF-T and CF-NS samples were checked by Fourier transform
represents the OH stretching vibrations and peaks appearing at 1634 cm-1 and 1380 cm-1 indicated
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1
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infrared (FTIR) spectrophotometer as shown in Fig. 2 (A), (B), respectively. The peak at 3423 cm-
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C=O stretching, and C–O stretching in carboxylic groups, and carboxylate moieties, respectively. The peaks of CF-NS are stronger than CF-T, which indicates that the oxidation treatment gave rise
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to an increase of oxygen-containing groups. The results of the current study are similar to the
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finding of Bao et al. [48] and Wang et al. [49]. FTIR spectrum for E.aerogenes is shown in Fig. 2 (C), which the peak at 3295 cm−1 indicates the N-H and O-H stretching vibration: polysaccharides,
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proteins. Peaks at 2959 cm−1 and 2926 cm−1 illustrate the CH3 asymmetric stretch: mainly lipids
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and the CH2 asymmetric stretch: mainly lipids, with little contribution from proteins, carbohydrates, and nucleic acids, respectively. The peak at 2854 cm−1 shows the CH3 symmetric
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stretch: largely proteins, with little contribution from lipids, carbohydrates, and nucleic acids. The peak at 2851 cm−1 displays the CH2 symmetric stretch: mainly lipids, with little contribution from proteins, carbohydrates, and nucleic acids. Peaks at 1759 cm−1 and 1384 cm−1 represent the ester C=O stretch: lipid, triglycerides and C-O symmetric stretch: amino acid side chains, fatty acids, respectively. Peaks at 1659 cm−1 and 1539 cm−1 demonstrate the amide (protein C=O stretching):
α helices, and the amide (protein N-H bend, C-N stretch): α helices. The peak at 1452 cm−1 shows the CH2 bending: lipids and the peak at 1237cm−1 indicates the PO-2 asymmetric stretching: mainly nucleic acids with little contribution from phospholipids. Peaks at 1150 cm−1 exhibit the CO-O-C
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asymmetric stretching: glycogen and nucleic acids, the peak at 1077 cm−1 depicts PO-2 asymmetric stretching: mainly nucleic acids with a little contribution from phospholipids and the peak at 948 cm−1 displays the C-N+-C stretch: nucleic acids. The present results have good agreement with the reports by Davis and Mauer [50]. FTIR spectrum for CF-NS– E.aerogenes is illustrated in Fig. 2 (D). Peak at 3289 cm−1 exhibits the combination of stretching vibration of N - H and O - H. The
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peaks at 1648 cm−1, 1532 cm−1, and 2358 cm−1 illustrate C = O stretching mode of amid group, N
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- H mode of amid group and C ≡ N mode of nitriles group, which indicates that E.aerogenes was
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successfully immobilized on the CF-NS by covalent binding.
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The surface morphology of CF supports using TEM and FESEM at different magnification
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scales are observed in Figs. 3 and 4, respectively. The TEM analysis shows that bacteria attach
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apparently on the CFs surfaces. As shown in Fig. 3-A4, the increment in the thickness of CFs after immobilization illustrate the presence of E. aerogenes on the surface of CFs. FESEM of CF
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samples in different magnification scales before and after cells immobilization is shown in Fig. 4. Figs. 4 A1, and A2 depict CF-0 before cells immobilization and Figs. 4 A3, and A4 indicate CF-0
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after cells immobilization. Figs. 4 B1 demonstrate CF-T before cells immobilization and Figs. 4 B2, B3, and B4 exhibit CF-T after cells immobilization. Figs. 4 C1 illustrate CF-NS before cells
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immobilization and Figs. 4, C2, C3, and C4 display CF-NS after cells immobilization. It can be concluded that the treatment of CF improved support properties and more cells could be immobilized on the surface of the CF-T. As shown in Fig. 4 immobilized cells loading amount is in the form of CF-T > CF-NS > CF-0. The surface morphology of PAC and GAC samples before
and after cells immobilization using FESEM at 1 µm scale is observed in Fig. 5. Figs. 5 A1 and A2 represent GAC before cells immobilization and Figs. 5 A3 and A4 after cells immobilization. Figs. 5 B1 and B2 illustrate PAC before cells immobilization and Figs. 5 B, and B4 show PAC
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after cells immobilization. For biofilm formation, a microorganism should encounter and attach a suitable surface and grow on the surface to produce a stable biofilm. The physical and chemical characteristics of the surface and the surrounding medium can affect biofilm formation [51]. Attachment of cells and development of biofilm on supports is related to the surface properties such as wettability,
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morphology and roughness, surface energy, electrical properties, and chemical composition of the
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supports [49]. The content of acidic functional groups and the moisture content value in CF-NS
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was higher than the corresponding values in CF-T. Total acidity and the moisture content values
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were %7 and 6.73 mmol/g for CF-NS and %2.6 and 3.49 mmol/g for CF-T, respectively. The
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present result shows that the wettability of CFs was increased with the increasing of hydrophilic
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functional groups on the CFs surface.
The mean mass of attached biomass on the support material was evaluated as the specific
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dry weight (g) of immobilized microorganisms on 0.01 g support at the end of fermentation, which
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was applied as a parameter to show the affinity between microorganism and support for biofilm formation or physical adsorption. The immobilized cells on CF-0, CF-T, and CF-NS, were 18.5,
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32.6, and 28.9 g/g, respectively. While the immobilized cells on PAC and GAC were 23.2 g/g and 13.9 g/g, respectively. Therefore, CF-T support was achieved high cell loading capacity. It is necessary for the support to immobilize high amount of biomass and provide conditions for efficient hydrogen production [52].
The HY, HPR, total volatile fatty acids (TVFAs) and ethanol yield, final pH, and glucose degradation efficiency (%) in the batch mode of operation for free and immobilized cells have been exhibited in Tables 1 and 2. As shown in Table 1 HY and HPR of free cells were 2.09 mol/mol
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glucose and 1.85 L/h. L, respectively. Hydrogen yield and hydrogen production rate of immobilized E. aerogenes on 0.2 mg/mL CF-0, CF-T, and CF-NS were 1.83, 2.56, and 2.11 mol/mol glucose, and 2.22, 2.48 and 2.10 L/h.L, respectively. The carbon fiber surface was functionalized to improve hydrophilicity and biocompatibility, however, a surface of very hydrophilic can also hinder immobilization of microorganism [48]. It seems microorganisms
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prefer to adhere to support surfaces with moderate hydrophilicity. Immobilization of cells on 0.2
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mg/mL of PAC and GAC resulted in HY of 2.13 and 1.33 mol/mol glucose and HPR of 2.51 and
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2.11 L/h.L, respectively. Comparison of various activated carbon as support materials (PAC and
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GAC) showed that PAC has higher HY and HPR. Powdered activated carbon has a relatively smaller particle size when compared with granular activated carbons and consequently, presents a
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larger surface to volume ratio. During the investigation of effect of support concentration, results
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showed that at lower concentration of all supports immobilized E. aerogenes was more effective,
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which may describe that mass transfer resistance was higher in high concentration of support material. Excessive amount of supports material may reduce contact between bacteria with
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substrate and nutrients [53]. Immobilization of E. aerogenes on GAC, PAC, and CFs resulted in the shorter lag phase compared to the control. The glucose degradation efficiency of immobilized
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cells of E. aerogenes on supports was lower than free cells. The glucose degradation efficiency when using CFs was 71-77%, which was lower compared to the control (95%). In the cases of PAC and GAC, the glucose degradation efficiency was 71-82%.
The main soluble metabolites measured during dark fermentation in the present study are butyric acid (HBu), propionic acid (HPr), acetic acid (HAc) and ethanol (EtOH), while similar results are published by Mohanraj et al. [54] and Lin et al. [55]. In free cells system, butyric acid
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is the primary VFAs product, followed by propionic acid and acetic acid. As shown in Table 2, HBu, HPr, and HAc for free E. aerogenes are 2907, 884, and 570 mg/ L, respectively. Using 0.2 mg/mL CF-T as support, HBu, HPr, and HAc are 1880, 374, and 336 mg/L, respectively. During dark fermentation of carbohydrates for hydrogen production, the pathways indicated that production of acetic and butyric acids releases hydrogen into the medium according to Eqs. (3)
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and (4). The overall equation for the production of propionic acid from glucose (Eq. (5)), showing
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that it involves consumption of hydrogen [56]. Ethanol as by-product of a metabolic pathway
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produces metabolites, which compete with hydrogen producing. When ethanol is produced, at least
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2mol of NAD+ are released. NAD+ can later on be recycled back to NADH in a reaction competing
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metabolites is unfavourable.
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with hydrogen production [10]. Therefore, presence of ethanol and propionic acid in end
(3)
C6H12O6 + 2H2O 2CH3COOH + 4H2 + 2CO2
(4)
C6H12O6 + 2H2 2CH3CH2COOH + 2H2O
(5)
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C6H12O6 CH3CH2CH2COOH + 2H2 + 2CO2
The concentration of 0.2 mg/mL CF-T was selected for the reusability study of
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immobilized cells of E. aerogenes in repeated batch experiments. As demonstrated in Table 3 after the sixth batch, HY decreased to 0.91 mol/mol glucose and HPR increased to 2.97 L/h.L. Table 3 shows a 1.34–1.61-fold enhancement in HPR from 1st to 6th batch when compared to the control. However, HY was decreased from 2.56 in 1st batch to 0.91 in 6th batch.
The cumulative hydrogen data are fitted to modified Gompertz model and the kinetic parameters are presented in Table 3. As illustrated in Table 4 and Fig. 6 the correlation coefficients (R2) of all the regressions are over 0.97, which illustrate that the modified Gompertz model fitted
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the data very well and estimated the progress of volumetric hydrogen production in the current study successfully. Table 4 reveals that the maximum hydrogen production potential (Hmax) for free E. aerogenes and immobilized E. aerogenes on 0.2 mg/mL CF-T by modified Gompertz model is 178 mL and 191 mL, respectively. Further, the aforesaid values are in good agreement with the experimental values of the present study.
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In the current study, treated and functionalized carbon fiber were used for the first time
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successfully for immobilization of E. aerogenes in dark fermentative hydrogen production.
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Comparison of three CFs (CF-0, CF-T, and CF-NS) as support for immobilization illustrated that
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CF-T provides higher biomass immobilization capacity, and consequently higher HY and HPR
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than CF-0 and CF-NS. The treated carbon fiber surface provides moderate hydrophilicity that is
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effective for cells immobilization. Immobilization of E. aerogenes on the surface of treated carbon fiber and functionalized carbon fiber is carried out by biofilm formation and covalent binding,
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respectively. The results show that HY of biofilm formation method is higher than HY of covalent binding method. The covalent binding provides durable attachment compared to biofilm
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formation, however, the strong binding may also inhibits cells movement and resulted in reduce their activity [7]. Comparison between activated carbon supports (PAC and GAC), showed that
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immobilization on PAC results in higher HY and HPR. Powdered activated carbon presents a smaller particle size (100 µm) when compared to GAC (2-2.5 mm) and provides a larger surface area and consequently, shows higher biomass immobilization capacity. Mohan et al. [35] employed GAC and PAC as supports to enhance dark fermentative hydrogen production and
indicated HY of GAC was higher in comparison to HY of PAC .The most relevant studies are shown in Table 5 and is concluded that the application of carbon supports is a promising method to improve hydrogen production from various substrates.
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3. Conclusions The current study has attempted to investigate biohydrogen production by immobilized E. aerogenes on the carbon fiber, surface modified carbon fiber, and granular and powdered activated carbon with an objective of enhancing biohydrogen production. Immobilization of E. aerogenes on CF, PAC and GAC has almost economic advantages such as reducing lag phase, generating
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faster of hydrogen production over a time period and supports are stable in the medium. Hydrogen
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yield of immobilized E. aerogenes on 0.2 mg/mL CF-0, CF-T, and CF-NS was 1.83, 2.56, and
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2.11 mol/mol glucose, respectively. While PAC and GAC resulted in HY of 2.13 and 1.33 mol/mol
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aerogenes to produce biohydrogen.
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glucose. The results indicate that CF may be used as a promising support for immobilization of E.
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Acknowledgements
The authors would like to express their gratitude to Iran National Science Foundation
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(Grant no. 93012717) for the financial support of the present study.
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[32] S.Y. Wu, C.Y. Chu, Y.C. Shen, Effect of calcium ions on biohydrogen production
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performance in a fluidized bed bioreactor with activated carbon-immobilized cells, Int. J. Hydrogen Energy. 37 (2012) 15496–15502.
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[37] J. Chang, K. Lee, P. Lin, Biohydrogen production with fxed-bed bioreactors, Int. J.
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[38] Z.-P. Zhang, J.-H. Tay, K.-Y. Show, R. Yan, D. Tee, D.T. Liang, D.-J. Lee, W.-J. Jiang, Biohydrogen production in a granular activated carbon anaerobic fluidized bed reactor, Int.
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TE
[39] S.N. Jamali, J. Jahim, W. Nor, R. Wan, P. Mohamed, Particle size variations of activated
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carbon on biofilm formation in thermophilic biohydrogen production from palm oil mill effluent, Energy Convers. Manag. 141 (2017) 354–366.
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[40] K. Lee, J. Wu, Y. Lo, Y. Lo, P. Lin, J. Chang, Anaerobic hydrogen production with an
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Schematic
diagram
A
CC
EP
TE
D
Figure1
M
A
N
U
SC RI PT
Figures and Tables
of
the
experimental
set
up
SC RI PT U N A M D TE EP
CC
Figure2. FTIR spectra of CF-T (A), CF-NS (B), Enterobacter aerogenes (C),
A
CF-NS- Enterobacter aerogenes (D)
SC RI PT U N A M D
Figure 3.TEM of CF-T -E. aerogenes conjugate in different magnification scales: 200 nm (A), 1
A
CC
EP
TE
µm (B) and (C), and 500 nm (D).
SC RI PT U N A M D TE
EP
Figure 4. FESEM image of CFs samples in different magnification scales before and after cells immobilization. A1 (5 µm), and A2 (100 µm) indicated CF-0 before and A3 (100 µm) and A4 (10
CC
µm) indicated CF-0 after cells immobilization. B1 (5 µm) indicated CF-T before and B2 (200 µm), B3 (100 µm), and B4 (10 µm) indicated CF-T after cells immobilization. C1 (5 µm) indicated CF-
A
NS before and C2 (200 µm), C3 (100 µm), and C4 (10 µm) indicated CF-NS after cells immobilization.
SC RI PT U N A
M
Figure 5. FESEM image of GAC and PAC in 1 µm scale. A1, A2, indicated GAC before and A3
A
CC
EP
TE
after cells immobilization.
D
and A4 after cells immobilization. B1 and B2 indicated PAC before and B3 and B4 indicated PAC
SC RI PT U N A M D TE
A
CC
EP
Fig. 6 .Kinetic analysis of batch experiments by modified Gompertz model
Table 1- Performance in the batch mode of operation Support
Type
(mg/mL)
glucose)
(L/h.L)
Control
0
2.09±0.46
PAC
0.2
CF- T
Final pH
1.85± 0.41
95
5.52±0.02
2.13±0.22
2.51±0.34
82
5.95±0.04
0.4
2.04±0.31
2.25±0.23
79
5.83±0.03
0.2
1.33±0.37
2.11±0.29
72
5.58±0.03
0.4
1.27±0.38
2.08±0.38
71
5.77±0.02
0.2
1.83±0.41
2.22±0.47
75
5.60±0.03
0.4
1.81±0.38
2.03±0.42
74
5.62±0.01
2.56±0.34
2.48±0.36
77
5.55±0.04
2.29±0.39
2.27±0.37
76
5.61±0.02
2.11±0.49
2.10±0.31
74
5.58±0.02
1.85±0.42
1.76±0.29
71
5.41±0.02
0.2
EP
0.2
TE
0.4 CF- NS
A
CC
0.4
U
N
A
M
CF-0
EG (%)
D
GAC
concentration HY (mol /mol HPR
SC RI PT
Support
Table 2- Produced end metabolites in the batch mode of operation Support concentration (mg/mL) End-metabolites (mg/L)
HAc
HPr
HBu
0
1331±19
570±13
884±26
2907±32
PAC
0.2
1706±27
237±8
395±20
3116±24
0.4
1897±14
541±11
412±9
2271±27
0.2
2036±21
396±14
305±39
1378±15
0.4
2252±35
452±17
660±16
2085±41
0.2
3659±28
421±22
647±33
3061±14
3090±37
499±18
440±9
2117±25
1025±26
336±25
374±17
1880±31
1627±18
532±23
475±32
2628±39
1033±23
679±29
253±13
1132±32
941±11
726±15
260±8
716±21
CF-0
M
GAC
N
Control
U
SC RI PT
EtOH
A
Support Type
0.4
0.2
EP
CF-NS
TE
0.4
D
0.2
CF-T
A
CC
0.4
Table 3- Reusability of immobilized E. aerogenes on 0.2 mg/mL CF-T HY
batch NO.
(mol
HPR
Yields of end metabolites (mg/L)
EG
/mol (L/h.L) HAc
HPr
HBu
1
2.56±0.34
2.48±0.36
1025±26
336±25
374±17
1880±31
77
5.55±0.04
2
2.14±0.31
2.53±0.22
2342.62
627.56
421.42
2155.45
76
5.12±0.05
3
1.99±0.29
2.57±0.21
1184.53
827.84
251.55
2574.02
72
5.08±0.03
4
1.56±0.26
2.64±0.39
1118.79
440.63
U
EtOH
1396.32
75
5.05±0.07
5
1.27±0.33
2.89±0.41
1454.91
322.68
320.15
2660.81
79
4.89±0.08
6
0.91±0.22
2.97±0.44
975.81
A
glucose)
Final pH
(%)
SC RI PT
Fermentation
881.25
315.25
3063.99
78
5.43±0.03
N
M D TE EP CC A
267.88
Table 4- Kinetic parameters of hydrogen production for free and immobilized E. aerogenes Modified Gompertz model Rmax (mL/h)
λ (h)
R2
Control
178
104
3.2
0.9984
0.2 mg/mL CF-0
154
72
1
0.9883
0.2 mg/mL CF-T
191
82
0.9
0.9855
0.2 mg/mL CF-NS
185
82
0.9
0.9786
0.2 mg/mL PAC
188
88
0.2 mg/mL GAC
150
N
U
SC RI PT
Hmax (mL)
M
Fermentation batch
0.9915
1.1
0.9893
A
1
A
CC
EP
TE
D
65
Table 5- Comparative studies on types of carbon support employed in dark fermentative hydrogen production Substrate
Support
Immobilization
type
method Physical adsorption 1.90-3.76 mol/mol sucrose
Seed sludge
Sucrose
AC
E.aerogenes
Glucose
MWCNT- Covalent binding COOH
Glucose
CF-NS
Covalent binding
Glucose
PAC
N
Biofilm
A
CF-T
Biofilm
Glucose
1.85-2.11 mol/mol glucose
Present
2.29-2.56 mol/mol glucose
TE
GAC
study Present study
2.04-2.13 mol/mol glucose
Present study
Biofilm
1.27-1.33 mol/mol glucose
Present study
AC
Granulation
1.42 mol/mol glucose
[9]
Glucose
CNT
Granulation
2.45 mol/mol glucose
[9]
Seed sludge
Glucose
AC
Granulation
1.57 mol/mol glucose
[26]
Seed sludge
Glucose
AC
Entrapment
0.87 mol/mol glucose
[26]
Seed sludge
Sucrose
AC
Entrapment
2.62 mol/mol sucrose
[33]
CC
Anaerobic sludge
Glucose
EP
E.aerogenes
[32] [27]
D
E.aerogenes
Glucose
M
E.aerogenes
Ref.
2.14 -2.2 mol/mol glucose
U
E.aerogenes
H2 yield
SC RI PT
Microorganism
A
Anaerobic sludge
Seed sludge
Sucrose
AC
Entrapment
1.82-4.98 mol/mol sucrose
Seed sludge
Chemical
GAC
Biofilm
1.28-2.38 mol/kg
wastewater
day
Chemical
PAC
Biofilm
1.22-1.57 mol/kg day
Biofilm
Seed sludge
Sucrose
CAC
Biofilm
Seed sludge
Glucose
GAC
Biofilm
Seed sludge
Glucose & xylose
GAC
Biofilm
Seed sludge
Sucrose
CAC
Biofilm
Sucrose
CAC
Seed sludge
Glucose
Seed sludge
Molasses
+
E.
A
homaechei 83
[36]
-
[37]
0.94-1.19 mol/mol glucose
[38]
0.31-1.01 mol/mol sugar
[39]
1.01-2.45 mol/mol sucrose
[40]
Biofilm
1.01-2.45 mol/mol sucrose
[28]
Biofilm
0.5-1.7 mol/mol glucose
[29]
GAC
Biofilm
3.47 mol/mol sucrose
[30]
AC
Biofilm
0.3-1.2 mol/mol glucose
[31]
TE
GAC
EP
2A
CC
HG6
Glucose
1.2-3.9 mol/mol sucrose
U
CAC
N
Sucrose
M
A
Seed sludge
E.cancerogeous
CODR- [35]
D
wastewater
Seed sludge
CODR- [35]
SC RI PT
Seed sludge
[34]