Short communication

Ethanol fermentation for main sugar components of brown-algae using various

yeasts

Sung-Mok Lee, Jae-Hwa Lee *

Department of Bioscience and Biotechnology, Silla University, Busan 617-736, Republic of Korea 1. Introduction

In the year 2008, greenhouse gas was already exceeding dangerously high levels of 450 ppm CO2, therefore the need for the development of alternative energy had already prompted many research projects around the world [1,2]. Many hope that renewable will be developed as an alternative to fossil fuels, with special attention being paid to bio-ethanol blending in gasoline [3– 5]. Using land plants for the manufacturing of alternative energy becomes a problem due to competition for food resources and the resulting higher price of cereals [6]. The growth rate of seaweed is superior to other biomass and it is harvested 3–4 times annually 3 off the coastal waters of Korea and 6 times annually in South-East Asia in the subtropic climates [7].

Seaweeds were classified according to their color schemes, like red, green, and brown algae, and these colors are related to the photobathic zone. Among the many types of seaweed, the brown algae that are most widely cultivated in Korea are Laminaria japonica and Undaria pinnatifida [7]. Brown seaweed contains about 30–67% carbohydrate by dry weight, which is the main component of polysaccharides such as alginate, laminaran, and mannitol [8,9]. The component concentration in the brown algae changed with seasonal changes [10,11]. Alginate has a high viscosity that forms cellular walls of brown algae that contain b-D- mannuronic acid and a-L-guluronic acid. In the January–March period, the biomass material had a much higher content of alginate, while the biomass had the lowest content of laminaran and mannitol in the same period; laminaran and mannitol comprised more of the content in the August–October period. Laminaran is mainly comprised of b-(1,3)-D-glucan with a small amount of b-(1,6) residues, in which the chain terminate including mannitol.

Due to the absence of various carbon sources, is expected that conversion of a biomass to bio ethanol will take place through biological conversion. But microorganisms have different preferred carbon sources [10,12], so the selection of microorganism needs to be made carefully in order to result in effective fermentation. The aim of this study was to determine the optimum yeast strain to use on substrates such as alginate, laminaran, and mannitol and the most representative brown algae L. japonica was used mixed composition for ethanol production.

2. Experimental

2.1. Microorganisms for ethanol fermentation

The microorganisms for ethanol fermentation were obtained from the Korea Culture Center of Microorganisms (http:// www.kccm.or.kr/) and Biological Resource Center (http:// www.brc.re.kr/main.aspx). We isolated 8 yeast strains, each affiliated with a different family, that might be suitable as an ethanol production microorganism, and each strain was utilized Journal of Industrial and Engineering Chemistry 18 (2012) 16–18 A R T I C L E I N F O

Article history:

Received 1 December 2010

Accepted 4 May 2011

Available online 10 November 2011

Keywords:

Yeast

Ethanol fermentation

Brown algae

Laminaria japonica

A B S T R A C T

The conversion of marine biomass to renewable energy was widely considered an alternative to fossil fuel, especially with regards to bio-ethanol blending in gasoline. Due to the absence of carbohydrate content, brown algae was expected to be possible organism for achieving ethanol production, but the selected microorganism needs to effectively ferment. We carried out experiments with 8 types of yeast affiliated with each different family for ethanol production and tested the effects of different carbon sources. Low concentrations of substrate were found to mostly increase the cell growth from all different substrates, but a significant increase in ethanol production was detected on the mannitol substrate. Saccharomyces cerevisiae (KCCM50550) was found to produce the highest result among all yeast strains, and ethanol production reached 2.59 g/L from 10.0 g/L of mannitol. A higher content of ethanol production in the fermentation was evident when the carbon source concentration increased.

2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.

* Corresponding author at: Department of Bioscience and Biotechnology, Silla University, Kwaebop-dong 1-1, Busan 617-736, Republic of Korea. Tel.: +82-51-999-****; fax: +82-51-999-****.

E-mail address: ac2l3p@r.postjobfree.com (J.-H. Lee).

Contents lists available at SciVerse ScienceDirect Journal of Industrial and Engineering Chemistry

jou r n al h o mep ag e: w ww .elsevier .co m /loc ate/jiec 1226-086X/$ – see front matter 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jiec.2011.11.097

for cell growth and ethanol fermentation experiments (Table 1). All the microorganisms were cultured for stock in an YPD broth

(glucose 20.0 g/L, peptone 20.0 g/L, yeast extract 10.0 g/L). The stock culture was maintained at 70 8C in 20% glycerol. Seed cultures for ethanol fermentation experiments utilized YPD broth, with culture conditions as follows: 150 rpm at 30 8C for 48 h in a shaking incubator.

2.2. Medium

Cell growth and ethanol production experiments with various main components of brown algae used defined media. All experiments were cultivated in a liquid nitrogen source medium containing 10.8 g/L (NH4)2SO4, 5.0 g/L H2KPO4, or 1.1 g/L MgSO4 7H2O that were combined with 10 g/L of alginate, laminaran, or mannitol carbon source, respectively. The brown-algae used for ethanol fermentation was carried out with L. japonica. L. japonica was obtained in a dry condition from the Bujeon market in Korea. The sample was finely grinded down with a ball miller (DW-BM5, Dongwon Scientific Co., Korea), and the sample was stored in an airtight container. All experiments were carried out in 300 mL flasks with 100 mL working medium, which consisted of 20 g/L milled L. japonica or defined media; the flasks were autoclaved at 120 8C for 15 min, inoculated with 3.0 mL of various yeasts after cooling to room temperature, and then they were capped with butyl rubber covers to create an anaerobic condition. The ethanol fermentation was subjected to 150 rpm at 30 8C via a shaking incubator with an initial pH of 7.0 without additional pH control during fermentation. The change of the ethanol product rate due to the carbon source concentration increase was carried out an experiment a five-fold increase to 50 g/L for the alginate, laminaran, and mannitol and 100 g/L for the L. japonica concen- tration. The nitrogen source was maintained.

2.3. Measured ethanol concentration and cell growth In order to measure the ethanol products, the supernatant of the culture broth that was pretreated at 12,000 rpm for 10 min was used in a gas chromatography (GC) analysis. The ethanol concentration was analyzed by a HP 5890 series gas chromatogra- phy instrument equipped with a flame-ionization detector and a HP-FFAP capillary column (Cross-Linked PEG-TPA 30 m/0.25 mm/ 0.25 mL). The inlet and detector temperatures were kept at 150 8C and 200 8C, respectively, and the oven temperature was pro- grammed as follows: 45 8C (2 min)/(1 8C/min)/50 8C (1 min)/

(20 8C/min)/90 8C (1 min)/(30 8C/min)/150 8C (1 min). The samples were measured with a split of 70:1 with a carrier gas used high purity nitrogen gas. Cell growth in the culture period was measured with a UV/Vis spectrophotometer (Optizen 2120UV, Mecacys Ltd., Korea) at 600 nm.

3. Results and discussion

The cell growth of various yeast strains in terms of fermentation with different carbon sources (alginate, laminaran, mannitol and L. japonica) are shown in Table 1. Ethanol fermentation was carried out in 300 mL flasks with 100 mL of working medium, prepared by exposure to 150 rpm for 96 h at 30 8C. All experiments were carried out in duplicate or triplicate. In all experiments, significant cell growth increases were detected; the maximum cell growth was observed in the Pichia stipitis (KCTC7228) and this strain was found to favor mannitol, as determined by optical density (O.D.) measurements at 600 nm which resulted in a value of approxi- mately 3.62. The range of O.D. measured for other carbon source conditions with the various yeast strains was 0.98–2.72. Generally, alginate resulted in lower increases of cell growth, but Pachysolen tannopilus (KCTC7229) and Kloeckeraspora osmophila

(KCCM50548) exhibited their highest increases with laminaran and mannitol. It is not easy to for the microorganism to consume the biomass on account of the uronic acid composition, and then the final product from alginate was 2-keto-3-deoxy glucoaldehyde via hydrolysis [13,14]; further reactions via enzymes needs to be done to use 2-keto-3-deoxy glucoaldehyde for ethanol fermenta- tion (2-keto-3-deoxy-6-phosphogluconate aldolase is a good candidate for fermentation). The possibility of using laminaran is high because it is comprised mainly of b-(1,3)-D-glucan, and an effective pretreatment of acid hydrolysis for the enhancement of cell growth and ethanol production has already been researched

[12]. A maximum cell growth was reached at an O.D. of 2.56 at 600 nm after 96 h for the Kluyveromyces marxianus (KCTC7150) in the laminaran medium. But the cell growth in most of the results was lower than the increases due to mannitol, which may be due to an imperfection hydrolysis from laminaran branching of (1,6)-b- glucosidic linkages [10]. The L. japonica containing medium was not detected by via the high particle content in the culture broth. Table 2 shows the results of ethanol production. Significant increases in ethanol production were detected only with mannitol, with a highest ethanol production for the Saccharomyces cerevisiae

(KCCM50550) of approximately 2.688 g/L and for the P. stipitis

(KCTC7228) an amount of 1.395 g/L was reached; the maximum ethanol production yield was 0.269 g per 1 g substrate. The maximum ethanol production exclusive of mannitol reached 0.404 g/L with the L. japonica medium and this accounts for approximately 28.96% of the mannitol fermentation in the same yeast strain condition. Others carbon sources insignificantly increased the fermentation, and the range of the amount of Table 1

Maximum cell growth using various substrates (alginate, laminaran and mannitol 10.0 g/L, Laminaria japonica 20.0 g/L) from the main composition of brown algae compared with various yeast strains.

Cell growth (600 nm) Substrate

Alginate Laminaran Mannitol

Pichia stipitis (KCTC7228) 1.92 1.72 3.62

Saccharomyces cerevisiae (KCCM50550) 1.82 1.80 2.40 Kluyveromyces marxianus (KCTC7150) 1.62 2.56 2.44

Debaryomyces occidentalis (KCTC7196) 1.03 1.16 2.39 Brettanomyces bruxellensis (KCCM11490) 0.98 1.48 1.52 Pachysolen tannopilus (KCTC7229) 2.72 1.55 1.64

Schizosaccharomyces pombe (KCCM11527) 1.68 1.49 1.82 Kloeckeraspora osmophila (KCCM50548) 2.12 1.53 1.22 Table 2

Maximum ethanol production using various substrates (alginate, laminaran and mannitol 10.0 g/L, Laminaria japonica 20.0 g/L) from the main composition of brown algae compared with various yeast strains.

Ethanol production (g/L) Substrate

Alginate Laminaran Mannitol L. japonica

Pichia stipitis (KCTC7228) 0.013 0.014 1.395 0.404 Saccharomyces cerevisiae

(KCCM50550)

0.039 0.016 2.688 0.061

Kluyveromyces marxianus

(KCTC7150)

0.028 0.039 0.030 0.035

Debaryomyces occidentalis.

(KCTC7196)

0.011 0.011 0.020 0.023

Brettanomyces bruxellensis

(KCCM11490)

0.013 0.011 0.273 0.012

Pachysolen tannopilus

(KCTC7229)

0.020 0.031 0.062 0.020

Schizosaccharomyces pombe

(KCCM11527)

0.062 0.017 0.047 0.057

Kloeckeraspora osmophila

(KCCM50548)

0.102 0.028 0.027 0.103

S.-M. Lee, J.-H. Lee / Journal of Industrial and Engineering Chemistry 18 (2012) 16–18 17 ethanol production from other carbon source and yeast strain conditions ranged from 0.011 to 0.273 g/L. Others previously reported the characterization of ethanol production from a Laminaria hyperborean extract comprised of laminaran and mannitol with four different microorganisms (Zymobacter palmae, Pichia angophorea, K. marxianus and Pacchysolen tannophilus)

[9,10]. All microorganisms could convert seaweed extract to ethanol in the report. But our experiments did not detect significant production of ethanol with K. marxianus and P. tannophilus. The result difference is due to the low concentration of carbon sources used, as most carbon sources could be used to increase cell growth. Therefore, a supplementary examination carried out on the effect of carbon source enhancement for ethanol production in our experiment.

In all the experiments, the carbon source concentrations increased the ethanol production by five-fold to 50 g/L in the defined carbon source media and to 100 g/L for the L. japonica concentration used in the complex media. The increased ethanol production with the high concentration carbon source is shown in Table 3. All ethanol production experiments were prepared by being subjected to 150 rpm for 7 day at 30 8C. The effects of the high concentration carbon sources for ethanol production were clearly confirmed for the laminaran and L. japonica medium. A maximal ethanol production of 10.86 g/L was reached in the fermentation of Debaryomyces occidentalis (KCTC7196) with the L. japonica medium; also, an ethanol production increase was detected in the mannitol medium. In the fermentation with mannitol, the conversion to ethanol was reduced, especially for S. cerevisiae (KCCM50550) and Brettanomyces bruxellensis

(KCCM11490), possibly because conversion was inhibited by the high concentration carbon source, so mannitol is not a general sugar, but a sugar alcohol. But, the fermentation of highly concentrated L. japonica may not lead to conversion inhibition due to lower contents of individual structure material. A high ethanol production with Schizosaccharomyces pombe

(KCCM11527) was detected on the laminaran medium, with a highest ethanol production approximately of 5.74 g/L, while K. osmophila (KCCM50548) reached 2.12 g/L. An increase of the ethanol production from L. japonica was observed on the P. stipitis

(KCTC7228) culture with a final concentration of 2.90 g/L, in contrast to insignificant increases on other substrates. 4. Conclusions

This objective of this study was to confirm the ethanol production characteristics of various yeast strains and the effect of the brown algae substrate on the ethanol production. The carbohydrates of the brown algae consisted of alginate, laminaran, and mannitol, and brown algae were utilized in the production of ethanol and biomass increases. Significant ethanol production was found only in the manntiol medium for low concentrations of the substrate on the S. cerevisiae (KCCM50550), with a maximum concentration of 2.688 g/L, and the other substrates did not convert biomass to ethanol but they did affect the cell growth increase. The overall maximal ethanol production reached was 10.86 g/L from 100 g/L of L. japonica with D. occidentalis (KCTC7196), followed by substrate concentration increases. Our results show that it is not easy to use this combination of organisms for conversion of biomass to ethanol. It was considered likely that a hydrolysis enzyme was needed for the effective conversion of laminaran to glucose; also, manntiol is in need of additional research to understand how it affects substrate inhibition with respect to ethanol fermentation.

Acknowledgement

This research was supported by a grant from the Marine Bioprocess Research Center of the Marine Biotechnology Program funded by the Ministry of Land, Transport and Maritime, Republic of Korea.

References

[1] P.M. Schenk, S.R. Thomas-Hall, E. Stephens, U.C. Marx, J.H. Mussgnug, C. Posten, O. Kruse, B. Hankamer, Bioenerg. Res. 1 (2008) 20.

[2] A. Singh, P.S. Nigam, J.D. Murphy, Bioresour. Technol. 102 (1) (2011) 10.

[3] W.S. Cho, Y.H. Chung, B.K. Kim, S.J. Suh, W.S. Koh, S.H. Choe, J. Plant Biotechnol. 34

(2) (2007) 111.

[4] J. Xu, M.H. Thomsen, A.B. Thomsen, Appl. Biochem. Biotechnol. 162 (1) (2010) 33.

[5] T.H. Kim, F. Taylor, K.B. Hicks, Bioresour. Technol. 99 (2008) 5694.

[6] R.P. John, G.S. Anisha, K.M. Nampoothiri, A. Pandey, Bioresour. Technol. 102 (1)

(2011) 186.

[7] J.I. Park, H.C. Woo, J.H. Lee, Korean Chem. Eng. Res. 46 (5) (2008) 833.

[8] S.M. Lee, J.H. Kim, H.Y. Cho, H. Joo, J.H. Lee, J. Korean Ind. Eng. Chem. 20 (5) (2009) 517.

[9] S.J. Horn, I.M. Aasen, K. Østgaard, J. Ind. Microbiol. Biotechnol. 24 (2000) 51.

[10] S.J. Horn, I.M. Aasen, K. Østgaard, J. Ind. Microbiol. Biotechnol. 25 (2000) 249.

[11] K. Østgaard, M. Indergaard, S. Markussen, S.H. Knutsen, A. Jensen, J. Appl. Phycol. 5

(1993) 333.

[12] S.M. Lee, J.H. Lee, Appl. Chem. Eng. 21 (2) (2010) 154.

[13] D.B. Choi, B.Y. Ryu, Y.L. Piao, S.K. Chio, B.W. Jo, W.S. Shin, H. Cho, J. Korean Ind. Eng. Chem. 14 (2008) 182.

[14] S.J. Horn, K. Østgaard, J. Appl. Phycol. 13 (2001) 143. Table 3

Maximum ethanol production using various concentrations of substrates (alginate, laminaran and mannitol 50.0 g/L, Laminaria japonica 100.0 g/L) with the main composition of brown algae compared with various yeast strains. Ethanol production (g/L) Substrate

Alginate Laminaran Mannitol L. japonica

Pichia stipitis (KCTC7228) 0.01 0.01 1.39 2.90

Saccharomyces cerevisiae

(KCCM50550)

0.03 0.18 0.04 0.06

Kluyveromyces marxianus

(KCTC7150)

0.10 0.24 0.10 0.14

Debaryomyces occidentalis

(KCTC7196)

0.03 0.11 1.54 10.86

Brettanomyces bruxellensis

(KCCM11490)

0.01 0.04 0.02 0.35

Pachysolen tannopilus

(KCTC7229)

0.03 0.21 0.06 0.37

Schizosaccharomyces pombe

(KCCM11527)

0.01 5.74 0.06 0.06

Kloeckeraspora osmophila

(KCCM50548)

0.01 2.12 0.07 0.57

18 S.-M. Lee, J.-H. Lee / Journal of Industrial and Engineering Chemistry 18 (2012) 16–18

Contact this candidate