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388

BIOSYNTHESIS OF ETHANOL WITH NEW IMMOBILIZED SYSTEMS

Ruzica JOVANOVIC-MALINOVSKA1, Elena VELICKOVA1, Slobodanka KUZMANOVA1, Eleonora WINKELHAUSEN1, Maja CVETKOVSKA2 & Christo TSVETANOV3

1Department of Biotechnology and Food Technology, Faculty of Technology and Metallurgy, University SS.Cyril and Methodius, Rudjer Boskovic 16, 1000 Skopje, R. Macedonia

2Department of Polymers, Faculty of Technology and Metallurgy, University SS.Cyril and Methodius, Rudjer Boskovic 16, 1000 Skopje, R. Macedonia

3Institute of Polymers, Bulgarian Academy of Sciences, Bl.103-A, Acad. G.Bonchev Str., 1113 Sofia, Bulgaria

ABSTRACT

Jovanovic-Malinovska R., Velickova E., Kuzmanova S., Winkelhausen E., Cvetkovska M. & Tsvetanov C. (2008): Biosynthesis of ethanol with new immobilized systems. Proceedings of the III Congress of Ecologists of the Republic of Macedonia with International Participation, 06-09.10.2007, Struga. Special issues of Macedonian Ecological Society, Vol. 8, Skopje.

With an increasing need for renewable fuels, growing attention has been devoted to ethanol, considered as a cleanest liquid fuel alternative to fossil fuels. Ethanol is a renewable, environmentally friendly fuel that is in-herently cleaner than gasoline. Recently, significant advances have been made towards the technology of ethanol production. The prospect included is microbiological fermentation with immobilized microorganisms. New biode-gradable hydrogels prepared by UV crosslinking were used for immobilization of Saccharomyces cerevisiae cells. These biodegradable hydrogels based on environmentally friendly polymer scaffolds were designed to meet the criteria specified for an immobilized system. Entrapment of yeast cells in a three-dimensional polymer matrix was achieved, and various properties of the polymer matrix as well as the activity of the yeast cells were studied. Eth-anol biosynthesis was achieved with PEO and HEC biodegradable hydrogels entrapping S. cerevisiae cell. These systems enabled the complete bioconversion of glucose. SEM images of microorganisms entrapped in these hydro-gels reveal a material in which microorganisms can attach and growth.

Key words: biofuels; bioethanol; biodegradable hydrogels; immobilization; batch fermentation.

ИЗВОД

Јовановиќ-Малиновска Р., Величкова Е., Кузманова С., Винкелхаузен Е., Цветковска М. и Цветанов Х. (2008): Биосинтеза на етанол со нови имобилизирани системи. Зборник на трудови од III Конгрес на еколозите на Македонија со меѓународно учество, 06-09.10.2007, Струга. Посебни изданија на Македонското еколошко друштво, Кн. 8, Скопје.

Потребата од обновливи горива доведе до зголемен интерес за етанолот, кој се смета за најчисто алтернативно течно гориво во однос на останатите горива. Етанолот лесно се обновува а воедно претставува гориво кое ја штити животната средина и е многу почисто од бензинот. Неодамна се направени значителни подобрувања во технологијата на производство на етанол, кои се однесуваат на микробиолошка ферментација со имобилизирани микроорганизми. Нови биоразградливи хидрогелови добиени со UV вмрежување беа употребени за имобилизација на клетки од квасецот Saccharomyces cerevisiae. Овие биоразградливи хидрогелови се добиени од еколошки полимерни мрежи и соодветсвуваат на барањата на еден имобилизиран систем. Беше постигнато заробување на квасочните клетки во тродимензионална полимерна матрица, на која и беа испитани својствата, а беше испитана и активноста на самите клетки. Етанолот беше синтетизиран со клетки на Saccharomyces cerevisiae заробени во PEO и HEC хидрогеловите. Системите овозможуваат комплетна биоконверзија на глукозата. SEM снимките на микроорганизмот заробен во овие хидрогелови ја покажуваат структурата на материјалот за кој микроорганизмот може да се прикачи и да расте.

Клучни зборови: биогорива; биоетанол; биоразградливи хидрогелови; имобилизација; дисконтинуирана ферментација.

Оригинален научен трудOriginal Scientific Article

Biosynthesis of ethanol with new immobilized systems

389Proceedings of the III Congress of Ecologists of Macedonia

Introduction

The vast majority of all fuels and carbon-con-taining chemicals are produced from fossil resourc-es. Studies predict that most kinds of fossil resourc-es will be depleted within the next century (BP Sta-tistical review). Furthermore the combustion of fos-sil fuels causes elevated levels of greenhouse gas-es in the atmosphere, which could possibly lead to global warming (Wigley, 2005). The continuous de-pletion of the fossil fuels reserves and consequent escalation in their prices has stimulated an exten-sive evaluation of alternative technologies and sub-strates to meet the global energy demand. As a re-sult, alternative sources of energy are increasingly being considered as potential substitutes for fossil fuels (Farrell et al., 2006). Using renewable sourc-es of energy will be a major contribution to reduc-ing net CO2 emissions, thereby helping to meet obli-gations under the Kyoto Protocol. Ethanol is consid-ered to be most promising as it is an alternative liq-uid fuel. Ethanol reduces air pollution and toxic gas emissions because it is a fuel which contains oxygen that is good for the environment. Combining ethanol with gasoline extensively reduces carbon monoxide that is responsible for as much as twenty percent of smog formation. And unlike other oxygenates, etha-nol is not harmful to the environment in the event of a fuel spill or leak.

The market for fuel ethanol will increase dra-matically in the near future because of EU and other legislation to promote the use of biofuels for trans-port. Ethanol constitutes 99% of all biofuels in the United States. The US Department of Energy has set a goal of replacing 30% of transportation fuel with bioethanol by 2025 (Ragauskas et al., 2006) and the European Union has established a goal of 5.75% bioethanol fuel by 2010 (Biofuel strategy). Thus, the energy and environmental implications of ethanol production are more important than ever.

As the fuel ethanol industry moves towards the future, producers continue to look for new ways of reducing operational costs, increasing efficien-cy, and surviving the unpredictable fluctuations in feedstock and fuel costs that can threaten profitabili-ty and the survival of their plants. New process tech-nologies such as microbial fermentation with immo-bilized microorganisms have been developed ena-bling the production of ethanol.

Yeast Saccharomyces cerevisiae is used all over the world as the major ethanol-producing mi-croorganism. The widespread development and ap-plication of immobilized cell technology has signif-icantly increased the necessity of profound knowl-edge of the behavior of the immobilized cells from both the physiological and the kinetic point of view. A number of industrial processes can benefit from this knowledge, since many of these immobilized

systems could favorably replace existing technolo-gies.

Immobilization leads to increased produc-tivities, simple separation of biocatalysts, long-term process stability, and the possibility of repeated or continuous use of the immobilized biocatalysts is given (Pajić-Lijaković et al. 2007). Thus immobili-zation can lead to higher profitability of biotechnical processes or even allows a process competitive to chemical modification. For immobilization of living cells the encapsulation or entrapment of the cells in a polymer matrix is the most preferred method.

The choice of the three-dimensional network matrix is very important for the good performance of an immobilized cell system. Hydrogels based on both natural and synthetic polymers have found var-ious applications. The high water content of hydro-gels contributes to their biocompatibility and thus the hydrogels can be used for controlled drug re-lease systems, as membranes for biosensors, scaf-folds in tissue engineering, and for many other ap-plications (Gupta and Ravi Kumar, 2001). Due to environmental issues, there is a growing interest in developing of hydrogels that guarantee biodegrada-bility. In recent years, hydrogels, both synthetic and natural, have become promising materials for bio-medical applications. Poly(ethylene oxide) hydro-gels are non-toxic and biocompatible materials that have been approved by the US Food and Drug Ad-ministration for biomedical application, and they meet all of the requirements for strength, absorb-ency and flexibility (Tsvetanov et al., 1998). It has been shown that poly(ethylene oxide) films can be successfully crosslinked in aqueous solutions by UV irradiation in the presence of photoinitiators such as benzophenone (Doycheva et al., 2004). The cellu-lose derivatives have received considerable atten-tion because of their water solubility and low costs. Therefore they have a very wide range of applica-tion in industry.

In this study biodegradable hydrogels based on environmentally friendly polymer scaffolds for yeast cells were prepared. Double-layer hybrid hy-drogels comprising hydrogel core with entrapped cells and outer hydrogel layer as barrier for cell leak-age and for mechanical strength were synthesized as well. Batch fermentation for biosynthesis of ethanol was performed. The applicability of the systems was estimated by some of the fermentation parameters.

Material and methods

Microoganism and chemicalsSaccharomyces cerevisiae, a commercial

grade baker’s yeast, with 32 % dry biomass was used. Polyethylene oxide (PEO or USA Polyox N12K) (Mn=1.106 g/mol) and hydroxyethylcellulose (HEC or Natrosol) (Mn=1.3.106 g/mol) were obtained from

Ruzica JOVANOVIC-MALINOVSKA et al.

390 Зборник на трудови од III Конгрес на еколозите од Македонија

Union Carbide Corp., sodium alginate with approxi-mately 70% G-block content was from Aldrich, and chitosan (medium molecular weight, Mn~4.105 g/mol) was from Fluka.. The (4-benzoylbenzyl) tri-methylammoniumchloride (BBTMAC) purchased from Aldrich was used as a photoinitiator, without further purification. All other chemicals used were purchased from commercial sources and were of an-alytical grade.

Preparation of hydrogelsAn aqueous solution of PEO (5% w/v) or

HEC (2% w/v) containing BBTMAC (2% in re-spect to the polymer solution) was poured into a te-flon Petri dish with a diameter of 2 cm forming a 2-3 mm thick layer. The polymers were UV irradiated in a Dimax light curing system 5000-EC equipped with 400 W metal halide flood lamp (input power = 93 mW/cm2), for 2 minutes (dose = 11.4 J/cm2). The PEO hydrogels including natural polymers (0.5% w/v sodium alginate, 0.5% w/v chitosan or 2% w/v HEC) were placed in a freezer at -400C for 2 hours and then UV irradiated in a temperature-controlled open chamber connected with cryostat apparatus.

Preparation of immobilized biocatalystsThe immobilized biocatalysts were formed

by adding yeast cells 5% w/v (corresponded to 1.6 g dry cells/Lmedium) to the mixture of polymers, followed by the same procedure as for preparation of pure hy-drogels was applied.

Characterization of the hydrogelsFor complete removal of the sol fraction,

the hydrogels were kept in distilled water that was changed frequently and then dried to constant mass under vacuum and weighed. The gel fraction (GF) was calculated as:

Gel fraction =

% (1)

The equilibrium degree of swelling (ES) was determined at room temperature. The hydrogels were equilibrated in distilled water or chloroform for at least 72 h, removed from the solvent, blotted with filter paper and weighed. They were then dried to constant mass under vacuum and weighed again. The ES is presented as grams of swollen gel sample per gram of dried gel sample:

Equilibrium swelling =

(2)Dynamic rheological measurements of the

hydrogels were performed on a Haake RheoStress 600 rheometer with a plate-plate system (20) and Peltier temperature controller. Shear storage modu-lus G′ and loss modulus G″ were measured in the

frequency range of 0.1-10 Hz at 25oC.Micrographs of cross-section and the interior

of biodegradable hydrogels loaded with Saccharo-myces cerevisiae cells were obtained by using a Jeol JSM-5510 scanning electron microscope (SEM). Composite hydrogels were quenched in liquid nitro-gen, freeze dried, and coated with gold in a Jeol JFC-1200 fine coater.

Fermentation with immobilized yeast cellsNutrient medium for reincubation had the

following composition (per litre): 10 g yeast ex-tract, 2 g KH2PO4, 1 g NaCl, 0.2 g CaCl2

.2H2O, 1.7 g MgSO4

.7H2O, 0.01 g FeCl3.6H2O, 2 g NH4Cl, and

20 g glucose. In the batch production of ethanol the glucose concentration was increased to 50 g/L. The media were autoclaved at 120oC for 15 min. The in-itial pH values of both media were 5. Immobilized biocatalysts were incubated in 50 ml nutrient medi-um placed in 250 ml Erlenmeyer flasks on a rota-ry shaker (150 rpm) at 28oC. After 20 hours of rein-cubation, the immobilized biocatalysts were washed several times in sterile distilled water and transferred into the medium for ethanol fermentation. The con-ditions for ethanol production were the same as for reincubation of the cells.

Analytical methodsSamples from the fermentation medium were

taken periodically and centrifuged. Cell concentra-tion was estimated turbidometrically at 620 nm, af-ter diluting the samples within the range of 0.05 to 0.5 units. One optical density unit corresponded to 0.3 mg dry cell mass/ml. Ethanol was analyzed by gas chromatography using a Varian CP 3800 with a capillary column. Nitrogen was used as a carrier gas with a flow rate of 30 ml/min. Isopropanol was used as an internal standard. Glucose concentration was determined by a dinitrosalycilic acid procedure of Miller.

Results and Discussion

Biodegradable hydrogels of poly(ethylene oxide) and its combination with natural polymers, alginate or chitosan, as well as hydroxyethylcellu-lose hydrogels, with or without additional crosslink-ing, were prepared by UV-irradiation of polymer so-lutions for 2 minutes in the presence of water sol-uble photoinitiator, (4-benzoylbenzyl) trimethylam-monium chloride (BBTMAC). The reaction showed high efficiency, and hydrogels with widely varying mechanical properties could be formed with this ap-proach. The size and shape of prepared PEO and HEC hydrogels are presented in Fig. 1.

In order to determine the efficiency of the UV-crosslinking, once prepared the gels were sub-mitted to a physico-chemical characterization. The



properties of the hydrogels are presented in Tab. 1.The hydrogels with shear storage modulus,

G′>200 Pa are considered to posses acceptable mechanical properties. The data for the PEO hydrogels with natural polymers, with rather high values of G′ lead to the conclusion that the mechanical properties are satisfactory. However, the high loss moduli and lower values of gel fractions refer to the necessity of changes in crosslinking procedure. The high G′ values of the HEC/BisAA-PEO and PEO/

alginate systems (Tab. 1) could be explained not only with the crosslinked PEO structure but also with the presence of natural polymers. As Kong et. al. (2003) stated, the stiff alginate chains contribute to high shear storage modulus. The additional layer increased the G′ values in comparison to the pure hydrogels.

Hydrogels prepared from PEO and HEC, without yeast cells, were cut and the morphology of the gels was examined by scanning electron

Fig. 1. PEO (a) and HEC (b) hydrogels (diameter 2 cm and height 3 mm).

Tab. 1. Efficiency of UV-crosslinking

System Gel composition (w/v %)

Gel fraction

(%)

Equilibrium swell-ing in

H2O CHCl3

Shear storage modulusG′ (Pa)

Loss modulusG″

(Pa)PEO 5 85 44 65 255 11HEC 2 83 24 / 1050 40HEC/BisAA-PEO 2/5 65 32 / 1298 190PEO/Ca-alginate 5/0.5 72 19 20 683 91PEO/GA-chitosan 5/0.5 60 37 61 270 37

Fig. 2. Scanning electron micrograph (SEM) of (a) PEO and (b) HEC hydrogels.



microscope (SEM). Fig. 2 shows a series of SEM images representing the cross section of the PEO and HEC gels without cells.

To check the yeast growth inside the polymer matrix, PEO and HEC hydrogels, with yeast cells were examined by scanning electron microscope (SEM) following the same procedure. The SEM im-age in Fig. 3 shows the proliferation of the yeast cells Saccharomyces cerevisiae entrapped inside the polymeric matrix after 24 hours of batch cultivation. Thus, this SEM photographs support our assumption of glucose utilization by the immobilized S. cerevi-siae cells.

The biosynthesis of ethanol by immobilized Saccharomyces cerevisiae, was used as a model re-action for testing the suitability of the hydrogels as immobilization matrices. For this purpose, these hy-

drogels have to be perfectly permeable to the sub-strate (glucose), to the other nutrients and to the product formed (ethanol). The suitability of these types of polymer supports used for immobilization on the ethanol production by immobilized Saccharo-myces cerevisiae cells are demonstrated in Fig. 4.

When the cells are entrapped in double-lay-er carriers, the diffusion of the substrate and prod-uct is restricted, which could lead to a deficien-cy of substrate within the gel. The substrate con-sumption after 24 hours of cultivation in PEO/Ca-alginate and PEO/GA-chitosan double-layer carri-ers were lower then other carriers, 52 and 51%, re-spectively. The fact that the glucose was consumed very slowly pointed to the diffusional limitations in the double-layer hydrogels. In addition, in fermenta-tion with PEO/GA-chitosan immobilized yeast, eth-

Fig. 3. Scanning electron micrograph (SEM) of PEO (a) and HEC (b) hydrogels loaded with Saccharomyces cerevisiae cells.

Fig. 4. Ethanol production of immobilized Saccharomyces cerevisiae in different biodegradable hydrogels



anol was not detected. The crosslinking of chitosan with glutharaldehyde caused inactivation of the liv-ing cells. The use of crosslinkers such as gluthar-aldehyde, are known to be relatively toxic, and the substrate was consumed for recovery of the immo-bilized cells.

The highest ethanol production coincid-ed with the fastest rate of glucose consumption ob-served (Fig. 5). Of all investigated hydrogels, PEO and HEC polymer carriers were considered as the most favorable. These systems enabled the complete bioconversion of glucose and highest ethanol con-centration (Tab. 2).

The fact that the glucose was consumed so fast in PEO and HEC biocatalityc systems gave ev-idence that there had been no diffusional limitations in these hydrogel carriers. These systems retained the yeast cells in the gels, allowed for the perfusion of the substrate and the product, and most important-ly, retained the activity of the cells.

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Fig. 5. Dynamics of glucose consumption in fermentation experiments using immobilized Saccharomyces cerevisiae in different biodegradable hydrogels.

Tab. 2. Process parameters of batch fermentation with immobilized Saccharomyces cerevisiae cells in different polymer carriers

SampleSubstrate con-

sumed(%)

Ethanol con-centration

(g/L)

Ethanol yield(g/g)

Ethanol pro-ductivity

(g/Lh)

Percentage of the theoretical yield

(%)

Time(h)

PEO 96.7 2.7 0.053 0.11 9.8 24HEC 96.8 3.3 0.066 0.14 12.9 24

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