The Use of Acrylamide Based Hydrogels in … Use of Acrylamide Based Hydrogels in Bioethanol Production ... and acrylamide/maleic ... presentation of possible copolymerization and

The Use of Acrylamide Based Hydrogels in Bioethanol Production

H. Nursevin ÖZTOP1, Dursun Saraydın2, A. Yasemin ÖZTOP3, Erdener Karadağ4, Yasemin IŞIKVER2 1Department of Biochemistry, Faculty of Science, Cumhuriyet University, Sivas, Turkey 2Department of Chemistry, Faculty of Science, Cumhuriyet University, Sivas, Turkey 3Department of Microbiology, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey 4Department of Chemistry, Faculty of Science, Adnan Menderes University, Aydın, Turkey

Bioethanol production is one of the most important technologies today because of the necessity to identify alternative energy sources. Fermentation carried out using immobilized microorganisms has many advantages compared with the use of free microorganisms. Immobilization has been shown to improve the feasibility of continuous processing, and increase cell stability, as well as lower overall costs of recovery, recycling, and downstream processing. Hydrogels can be used for this purpose as solid carriers. A novel composite hydrogels, radiation crosslinked 2-hydroxyethyl methacrylate/acrylamide (HEMA/AAm) and acrylamide/maleic acid (AAm/MA) copolymers and chemically crosslinked acrylamide/sodium acrylate (AAm/SA) copolymers were prepared and used for the immobilization of yeast cells. Saccharomyces cerevisiae immobilized hydrogels were successfully developed and applied to bioethanol fermentation and they have potential in industrial applications of the bioethanol production process.

Keywords: Bioethanol, hydrogel, acrylamide, immobilization, S. cerevisiae

1. Introduction

Biofuels are attracting interest today. Bioethanol production is one of the most important technologies as an alternative energy sources. Bioethanol is a nontoxic, biodegradable and renewable resource as biofuel. Bioethanol is produced using free or immobilized cells. Immobilized cell systems have many advantages over free cell systems. These advantages are cell stability, enhance fermentation productivity, repeated cycles of biotechnological processes, and improve the feasibility of continuous processing. Various support materials have been used for bioethanol fermentation with immobilized cells. These materials can be -alumina, ceramic, sodium alginate gel, metal ion-loaded collagen fiber, polymeric supports [1-5]. Hydrogels are polymeric networks which absorb and retain water without dissolving. This property makes them interesting materials as carriers for immobilization of bioactive compounds as alternatives to others successfully used. In our articles radiation induced acrylamide based hydrogels have been studied in adsorption of proteins, pharmaceuticals, cationic dyes and heavy metal ions [6-11]. Acrylamide based hydrogels were used immobilization and production of bioethanol in our other works [12-14]. This chapter is a mini-review of our works for bioethanol production.

2. Preparation and Characterization of Hydrogels

For bioethanol production by immobilized yeast cells (Saccharomyces cerevisiae), radiation crosslinked 2-hydroxyethyl methacrylate/acrylamide (HEMA/AAm) and acrylamide/maleic acid (AAm/MA) copolymers and chemically crosslinked acrylamide/sodium acrylate (AAm/SA) copolymers were prepared. Swelling and diffusion experiments were done for characterization of these hydrogels. Used monomer and comonomers are shown in Table 1

2.1 HEMA/AAm Hydrogels

Five grams of AAm and various amounts of HEMA (0, 1, 2, 3, and 5 ml) were mixed in distilled water and placed in PVC straws of 3-mm diameter and irradiated. A dose of 3 kGy at air temperature in irradiator was applied at a fixed rate of 6 kGy h−1. The radiation crosslinked HEMA/AAm copolymers obtained in long cylindrical shapes were cut into pieces of 4–5 mm long, dried in air and under vacuum. When monomers of HEMA and AAm in aqueous solutions have been irradiated with ionization rays such as -rays, free radicals are generated in the aqueous solutions. Random reactions of these radicals with monomers lead to the formation of copolymers of HEMA/AAm. When the irradiation dose was increased beyond a certain value, the polymer chains crosslink and then a gel is obtained. A schematic presentation of possible copolymerization and crosslinking reaction mechanisms between HEMA and AAm monomers is shown in Fig. 1.

Polymer science: research advances, practical applications and educational aspects (A. Méndez-Vilas; A. Solano, Eds.) _______________________________________________________________________________________________

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2.2 AAm/MA Hydrogels

Various amounts of MA (0, 20, 30, 40, 50, 60 mg) were mixed with 1 g of AAm, dissolved in 1 ml distilled water, placed in PVC straws of 3 mm diameter and irradiated to 2.60, 3.73, 4.65, 5.20, and 5.71 kGy in air at ambient temperature in a g irradiator with the dose rate of 0.72 kGy h−1. The radiation crosslinked AAm/MA copolymers obtained in long cylindrical shapes were cut into pieces of 4–5 mm length, and dried in air at room temperature and then under vacuum in a vacuum oven at 1 mmHg for one week. During polymerization and crosslinking reactions, all monomers reacted together by applied -radiation. The radiation technique was used for the sterilization of hydrogel systems at the same time. There was no monomer (such as toxic acrylamide) remaining at the end the polymerization and crosslinking reactions. A schematic presentation of possible copolymerization and crosslinking reaction mechanisms between AAm and MA monomers is shown in Fig. 2. Table 1 Some properties of monomer and comonomers

Monomer Symbol Chemical structure Molar mass Manufacturer

Acrylamide AAm

CC

H

O

C

HH

NH2

116.07 B.D.H. (Poole-UK)

2-hydroxyethyl methacrylate

HEMA 130.14 B.D.H. (Poole-UK)

Maleic acid MA 71,08 B.D.H. (Poole-UK)

Sodium acrylate SA 94.04 B.D.H. (Poole-UK)

2.3 AAm/SA Hydrogels

AAm/SA hydrogels were prepared by free-radical crosslinking copolymerization of AAm and SA with a small amount of different type crosslinkers in aqueous solution. SA was used as the ionizable comonomer. Ammonium persulphate and N,N,N’,N’-tetramethylenediamine (TEMED) were the initiator and the accelerator, respectively. To obtain AAm/SA hydrogels, 1 g of AAm, various amounts of SA (0, 10, 20, 30, and 40 mg), and 0.25 ml ammonium persulphate (1%), 0.15 ml TEMED (1%) and a crosslinker (1% 1,4-butandiol dimethacrylate; B or ethylene glycol dimethacrylate; E or N,N’ methylenebisacrylamide; N or trimethylolpropan triacrylate; T) were mixed in distilled water and placed in PVC straws of 3 mm diameter. A gel formed after 2 h of reaction at ambient temperature. After 24 h, the hydrogel rods containing different types of crosslinkers and various amounts of SA, were cut into 4–5 mm in length and washed with distilled water and dried in air and vacuum. A schematic presentation of possible copolymerization and crosslinking reaction mechanisms between AAm and SA monomers is shown in Fig. 3.


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+

water radical

AAm

+

HEMA

-rays

-rays

-rays

radical formation

initiation

copolymerization

crosslinking-rays

Fig. 1 The possible copolymerization mechanism of HEMA with AAm.

Fig. 2 The possible copolymerization mechanism of AAm with MA


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Fig. 3 The possible copolymerization mechanism of AAm with SA.

2.4 Swelling and Diffusion

For the swelling experiments HEMA/AAm hydrogels which prepared with 5.2 kGY irradiated dose, AAm/MA hydrogels which prepared with 5.2 kGY irradiated dose, AAm/SA hydrogels which prepared with N crosslinker were used. The hydrogels were swelled in the nutrient medium of yeast cells at 30 °C to reveal the parameters of swelling and diffusion. Swollen gels removed from the water bath at regular time intervals were dried superficially with filter paper, weighed, and placed in the same bath. The radii of cylindrical swollen gels were measured with a micrometer. In the dry state, hydrogels were hard and glassy, but in the swollen state, gels were soft and easy to handle. On swelling the hydrogels retained their integrity. The percentage of swelling (S %) of the hydrogels in the nutrient medium of the cells was calculated from the following relationship:

100% xmo

momtS

(1)

Here mt is the mass of swollen gel at time t and mo is the mass of the dry gel. The swelling curve of these hydrogels in the nutrient medium of the cells was plotted [12-14]. The swelling curves for AAm/ MA hydrogels irradiated to 5.2 kGy are presented Fig. 4 as an example. AAm/MA hydrogels and SA content in the AAm/SA hydrogels [12-14].

S %

Swelling time / minute

Fig. 4 Change in S% of AAm/MA hydrogels irradiated at 5.2 kGy with time (△, 0 mg MA; , 20 mg MA; ⵔ, 30 mg MA; ▲, 40 mg MA; , 50 mg MA; , 60 mg MA)


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The values of S% increased with time but reached a constant value after a certain point. This value of swelling may be called equilibrium swelling degree (Seq %). These values are listed in Table 2. Table 2 The variation of the values of swelling degree of the hydrogels in the nutrient

HEMA/AAm AAm/MA AAm/SA AAm content

mol % Seq %

MA content mg

Seq %

SA content mg

Seq %

0.00 34 0 885 0 759 63.18 436 20 1190 10 1506 74.17 625 30 1255 20 1630 81.09 797 40 1290 30 1672 89.58 949 50 1435 40 1804 100.00 1000 60 1455

For non-ionic hydrogels swelling is controlled by the hydrophilicity of the monomers or polymers. The hydrophilicity of AAm is higher than that of HEMA. As shown in Table 2, the value of the equilibrium swelling of the radiation crosslinked HEMA homopolymer (Seq % = 34) is approximately 30-fold lower than that of the radiation crosslinked HEMA/AAm homopolymer (Seq% = 1000). On the other hand, the equilibrium swelling of HEMA hydrogel increased with the addition of AAm monomer. At the same time, the equilibrium swelling of the hydrogels increased with an increase of AAm content in the HEMA/AAm hydrogels. The hydrophilicity of AAm/MA is higher than that of AAm due to ionization of carboxylic group from MA. As shown in Table 2, the values of the equilibrium swelling degree of the radiation crosslinked AAm (Seq % = 885) is approximately 2-fold lower than those of the radiation crosslinked AAm/MA hydrogels (Seq% = 1455). The equilibrium swelling of AAm hydrogels increased with the addition of MA monomer. At the same time, the equilibrium swelling of the hydrogels decreased with an increase of adsorbed irradiation dose (Table 3). Table 3 The variation in the equilibrium swelling degree of AAm/MA hydrogels with irradiation dose in the nutrient medium (MA content: 40 mg)

Dose (kGy) Seq % 2.60 1548 3.73 1522 4.65 1319 5.20 1290 5.71 1178

The hydrophilicity of AAm/SA is higher than that of AAm. As shown in Table 2, the values of the equilibrium swelling degree of the AAm (Seq % = 759) is approximately 2-fold lower than those of the AAm/SA hydrogels (Seq% = 1804). The equilibrium swelling of AAm hydrogels increased with the addition of SA monomer. The biggest swelling degree of various crosslinker containing AAm/SA hydrogel was the one containing N crosslinker (Table 4). Table 4 The variation of the values of swelling degree of the in AAm/SA hydrogels with the different type of crosslinker (amount of SA 30 mg)

Crosslinkers Seq % N 1672 E 1378 B 1215 T 844

Environmental conditions such as pH, ionic strength, and composition of the surrounding fluids affected the swelling nature of the hydrogels. Thus, the surrounding fluids formed the difference in the swelling degrees of the hydrogels. The study of diffusion phenomena in hydrogels and fluids is of value in that it clarifies polymer behavior. The following equation was used to determine the nature of diffusion nutrient medium into the hydrogels [15, 16]: F=ktn (2) In this equation F denotes the amount of penetrant fraction at time t; k is a constant incorporating the characteristics of the polymeric network system and the penetrant; n is the diffusional exponent, which is the indicative of the transport mechanism. The diffusion coefficient (D) of the cylindrical the hydrogels was calculated from Eq. (3) [15]:


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2/12/1

24 t

l

DF

(3)

Here D (in cm2 s−1) is the apparent diffusion coefficient for the transport of the penetrant into the gel, t is the time and l is the radius of cylindrical polymer sample. The values of the diffusion parameters of hydrogels are listed in Tables 5. Table 5 The values of diffusion parameters of the hydrogels in nutrient medium

HEMA/AAm AAm/MA AAm/SA AAm mol %

k x102 n Dx107 cm2s-1

MA mg

k x102 n Dx107 cm2s-1

SA mg

k x102 n Dx107 cm2s-1

0.00 10.59 0.46 5.6 0 2.84 0.60 6.97 0 3.14 0.59 9.42 63.18 2.62 0.62 6.6 20 1.95 0.66 4.72 10 0.92 0.68 4.23 74.17 2.53 0.66 9.2 30 1.97 0.64 5.69 20 1.18 0.67 5.92 81.09 2.36 0.68 12.2 40 1.70 0.66 6.10 30 1.04 0.68 5.49 89.58 2.57 0.63 9.5 50 1.90 0.65 6.62 40 0.88 0.69 5.29

100.00 2.27 0.65 10.4 60 1.75 0.66 5.63 The values of n were found to be between 0.50 and 1 (Table 5) and hence the diffusion of the fluids into the hydrogels was taken to be non-Fickian in character. This is generally explained as being a consequence of the slow relaxation rate of the hydrogel [15]. The values of the diffusion coefficient varied from 4.23 x 10-7 to 12.2 x 10-7. These results are accordance with the swelling results of the hydrogels.

3. Immobilization of yeast cells onto hydrogels and production of bioethanol

3.1 Preparation of the cells

Yeast cells were precultured for 48 h at 28 °C in an aqueous solution containing 1% glucose, 0.1% molasses, 0.5% peptone, 0.3% yeast extract and 0.3% malt extract (pH 4.8).

3.2 Immobilization of yeast cells onto hydrogels

Dry hydrogels weighing 0.1 g were sterilized with and then immersed in a mixture of precultured yeast cells (5 mg wet wt.) and nutrient medium (10 ml). The composition of the nutrient medium was of 12% glucose, 1% molasses, 0.15% yeast extract, 0.25% NH4Cl, 0.1% NaCl, 0.001% CaCl2 and 0.3% lactic acid (pH 4.8). The suspension was incubated in a rotary shaker for 72 h at 30 °C, and the nutrient medium was renewed every 24 h. Light microscope photographs of S. cerevisiae cells that interact with the AAm/SA hydrogels are shown in Fig. 5. A structural defect such as false and budding structural fiber formation in S. cerevisiae cells has not occurred that interact with the hydrogel.

Fig. 5 Light microscope photographs of S. cerevisiae cells that interact with the AAm/SA hydrogel

3.3. Fermentation and bioethanol analysis

Immobilized yeast cells were immersed in fresh nutrient medium and fermented by incubation at 30 °C under gentle rotary shaking. The concentration of bioethanol produced was determined using alcohol dehydrogenase [17]. Bioethanol production curves for AAm/SA hydrogels crosslinked with N are presented Fig.6 as an example. Production

40 x 100 x


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of bioethanol increased with time but reached a constant value after 40–80 min. On the other hand, the amount of bioethanol produced increased with an increase in the AAm content in the HEMA/AAm hydrogels, MA content in the AAm/MA hydrogels and SA content in the AAm/SA hydrogels [12-14].

C /

g (L

g g

el)-1

Incubation time /minute Fig. 6 Bioethanol production curves of immobilized S. cerevisiae in the different amount of SA containing N crosslinked AAm/SA hydrogels (;0 mg SA, ⵔ;10mg SA, ;20mg SA, ;30mg SA, ▲;40mg SA).

For production of bioethanol by immobilized cells, the following second order kinetics relationship can be written;

WtQC

t (4)

Where C is the concentration of bioethanol at time t, W=1/Cmax is the inverse of the maximum or equilibrium concentration of the producing bioethanol, Q=1/(dC/dt)o, is the reciprocal of the initial bioethanol production rate (rP) of the gel. The values of initial production rate and maximum concentration of bioethanol were calculated from the slope an intersection of the lines and, are presented in Table 6. Table 6 The values of bioethanol production parameters of the hydrogels

HEMA/AAm AAm/MA AAm/SA AAm mol %

Cmax g (L g jel)-1

rP g (L g gel h)-1

MA mg

Cmax g (L g gel)-1

rP g (L g gel h)-1

SA mg

Cmax g (L g gel)-1

rP g (L g gel h)-1

0.00 0.000 0.00 0 17.00 66.37 0 10.1 17.4 63.18 5.146 7.68 20 17.15 311.59 10 20.4 16.2 74.17 6.932 13.68 30 17.35 312.00 20 20.0 34.2 81.09 7.455 22.20 40 18.21 313.15 30 21.5 49.2 89.58 8.755 27.90 50 18.92 341.22 40 23.8 33.6

100.00 18.201 66.36 60 19.60 730.70 Table 6 shows that the parameters of the maximum concentration of the producing bioethanol and the initial bioethanol production rate of the radiation induced hydrogels increased with the increase in the AAm content in the HEMA/AAm hydrogels, MA content in the AAm/MA hydrogels and SA content in the AAm/SA hydrogels. These results are parallel to the results of the equilibrium swelling of the hydrogels. When the values of equilibrium swelling of the hydrogels increased, the production of bioethanol also increased. Fig. 7 shows that the parameter of maximum concentration of the production ethanol is decreased with an increase in the irradiation dose.


550

Cm

ax /

g (

L g

gel

)-1

Irradiation dose /kGy

Fig. 7 The variation of the maximum producing of ethanol of the AAm/MA hydrogels with irradiation doses (MA content: 40 mg) Fig. 8 shows that the parameter of maximum concentration of the production bioethanol is increased with the order N ˃ E ˃ B ˃ T crosslinkers.

C

max

/ g

(L

g g

el)-1

Crosslinker type

Fig. 8 The variation of the maximum producing of ethanol of the AAm/SA hydrogels with the various crosslinker

4. Conclusion

With the study it is aimed to find the best carrier for immobilization of yeast cells and production of bioethanol by these cells. Bioethanol production is increased with increase in the amount of AAm in the HEMA/AAm hydrogel. When the content of MA in the AAm/MA hydrogel increased the production of bioethanol also increased. On the other hand, an increase in irradiation dose decreased the production of bioethanol. Bioethanol production is increased with increase in the content of SA in the AAm/SA hydrogel. Also the highest production was achieved with N crosslinker between crosslinkers. Fig. 9 shows swelling of the hydrogels, and Fig. 10 shows bioethanol production of the hydrogels.

0,00

5,00

10,00

15,00

20,00

25,00

2,60 3,73 4,65 5,20 5,71

0

5

10

15

20

25

N E B T


551

Seq

%

Fig. 9 The swelling degree of the hydrogels

Cm

ax /

g (L

g g

el)-1

Fig. 10 Bioethanol production of the hydrogels As shown in Fig. 9 and Fig. 10 bioethanol production was strongly dependent on the equilibrium swelling of the hydrogels. All factors that increase the swelling have increased bioethanol production. At the early stage of immobilization, the hydrogels swelled with the nutrient medium of cells as much as possible. This nutrient medium inside of the hydrogels must be replaced with yeast cells during the immobilization process. The hydrophilic groups of the hydrogels were increased with the increasing hydrophilic content. Thus, the swelling of the hydrogels was increased. The higher swelling of the hydrogels permitted the presence of more nutrient medium and cells inside of the hydrogel. Some yeast cells adsorbed onto the surface of the hydrogel and the adsorbed yeast cells infiltrated into the hydrogel through the small pores. Then, the yeast cells inside the hydrogel multiplied. The increase in the volume caused by the multiplying yeast cells resulted in the extension of the pores in the hydrogels. In this way, the production of bioethanol was increased. The maximum amount of bioethanol produced with crosslinkers in order of N > E > B > T. It can be deduced that N crosslinker provides maximum swelling resulting in maximum amount on immobilization of S. cerevisiae. Hence, the production of bioethanol is greater than the other used crosslinkers. The crosslinker has great effect on pore size. For instance, T has three functional groups and cause more tightly network structure resulting in decrease in the pore size of AAm/SA hydrogels. Consequently, the swelling is low as well as the immobilization of yeast cells. Other used crosslinkers E and B make the pore size between N and T. And these was reflected on swelling behavior and immobilization of yeast cell at same magnitude which caused in the amount of produced bioethanol in

0

250

500

750

1000

1250

1500

1750

2000

HEMA AAm HEMA/AAm AAm/MA AAm/SA

0,00

5,00

10,00

15,00

20,00

25,00

HEMA AAm HEMA/AAm AAm/MA AAm/SA


552

between N and T used crosslinkers. The elasticity of hydrogel network gives great advantages for the extent of immobilized yeast cell and for the production of bioethanol. In general, it can be said that these hydrogels could also be used for the immobilization of other cells due to their soft and elastic nature for the production of some bioactive species.

References

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[7] Karadağ E, Saraydin D, Öztop HN, Güven O. Adsorption of bovine serum albumin to acrylamide/itaconic acid hydrogels. Polym. Adv. Technol. 1994; 5: 664-8.

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[10] Öztop HN, Hepokur C, Saraydin D. Acrylamide-sepiolite based composite hydrogels for immobilization of invertase. Journal of Food Science. 2009; 74: 45-49.

[11] Öztop HN, Hepokur C, Saraydin D. Poly(acrylamide/maleic acid)-sepiolite composite hydrogels for immobilization of invertase. Polym. Bull. 2010; 64: 27-40.

[12] Oztop, H N, Oztop A Y, Isıkver Y, Saraydin D. Immobilization of Saccharomyces cerevisiae on to radiation crosslinked HEMA/AAm hydrogels for production of ethyl alcohol. Process Biochemistry. 2002; 37: 651–57.

[13] Saraydin D, Oztop H N, Karadag E, Oztop A Y, Isıkver Y, Guven O. The use of immobilized Saccharomyces cerevisiae on radiation crosslinked acrylamide–maleic acid hydrogel carriers for production of ethyl alcohol. Process Biochemistry. 2002; 37: 1351–57.

[14] Oztop H N, Oztop A Y, Karadag E, Isıkver Y, Saraydin D. Immobilization of Saccharomyces cerevisiae on to acrylamide–sodium acrylate hydrogels for production of ethyl alcohol. Enzyme and Microbial Technology. 2003; 32: 114–19.

[15] Rosiak JM, Ulanski P. Synthesis of hydrogels by irradiation of polymers in aqueous solution. Radiat. Phys. Chem. 1999; 55: 139-51.

[16] Vervoort L, Van den Mooter G, Augustijns P, KingetR. Inulin hydrogels. I. Dynamic and equilibrium swelling properties. Int. J Pharm. 1998; 172: 127-35.

[17] Bernt E. Gutman I. Ethanol determination with alcohol dehydrogenase and NAD. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis. Florida: Verlag Chimie International; 1974.p.1499-502.


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The Use of Acrylamide Based Hydrogels in … Use of Acrylamide Based Hydrogels in Bioethanol Production ... and acrylamide/maleic ... presentation of possible copolymerization and