A Facile Pretreatment Method for Rice Straw using Electron Beam Irradiation and 4-methylmorpholine-N-oxide Solution

전자선 조사와 4-메틸모포린-N-옥사이드 용액을 이용한 볏짚의 전처리 방법

이병민, 이진영, 강필현, 전준표*

한국원자력연구원공업환경연구부

Received: July 15, 2014 / Revised: August 18, 2014 / Accepted: September 16, 2014

서 론

세계적으로화석연료의사용은지구온난화문제등의환 경문제를초래함과동시에화석연료의고갈로인하여지난

2005

⁽

⁾

^[17].

(lignocellulose)

^.

(cellulose),

(hemicellulose),

^(lignin)

⁽

^-1,4- glucosidic bond)

^(glucose)

(semi-crystalline polymer)

[18].

^,

(pentose),

^(hexose),

(acetylated sugar)

(amorphous polymer)

(phenyl propane units: p-coumaryl, coniferyl and sinapyl alcohol)

C-C, C-O-C

^{[6, 10].}

(glucose),

(xylose)

⁽

로오스와헤미셀룰로오스

⁾

.

^.

향상에있다

^[13].

를이용하는화학적전처리방법

^{[19, 30],}

^[9],

^[28]

[8, 14, 27]

A Facile Pretreatment Method for Rice Straw using Electron Beam Irradiation and 4-methylmorpholine- N-oxide Solution

Byoung-Min Lee, Jin-Young Lee, Phil-Hyun Kang, and Joon-Pyo Jeun*

Research Division for Industry & Environment, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea In this study, a facile two-step pretreatment method was investigated for producing fermentable sugars. Rice straw was pre- treated using electron beam irradiation (EBI) and 4-methylmorpholine-N-oxide (NMMO) prior to enzymatic hydrolysis. In the first stage, the EBI on the rice straw was carried out at various doses (100, 300, 500 kGy) and then, irradiated rice straw was stirred with NMMO solution at 120

C for 1 h for the second stage. The pretreated rice straw was hydrolyzed by cellulase 1.5 L (70 FPU/ml) and Novozyme-188 (40 CbU/ml) at 50

C for 24, 48, and 72 h. A sugar yield of 83.8% was obtained from the pre- treated rice straw after 72 h of enzymatic hydrolysis. Also, FTIR and XRD results indicate that the pretreatment of the rice straw was effective due to the synergic effects of the two-step pretreatment. In conclusion, rice straw might be a potential substrate for bioethanol production by yeast fermentation.

Keywords: Biomass, pretreatment, lignocellulose, electron beam, rice straw

*Corresponding author

Tel: +82-62-570-3063, Fax: +82-62-570-3098 E-mail: [email protected]

© 2015, The Korean Society for Microbiology and Biotechnology

되고있다

^.

4-methylmorpholine- N-oxide

⁽

^NMMO)

Lyocell process

^{. Lyocell} process

^NMMO

^N-O

인공정이다

^[7].

^NMMO

의전처리법이보고되고있으며셀룰로오스와헤미셀룰로오 스에는영향을주지않고리스노셀룰로오스의결정화도를 감소시키며전처리후

^NMMO

^98%

려져있다

^{. NMMO}

(non-solvent)

(phase inversion)

[11, 21, 25].

나

^{, NMMO}

NMMO

17%

NMMO

이

^83%

^17%

경우낮은용해도를갖게되고주로셀룰로오스가팽윤되는 현상이나타난다고알려져있다

^[5].

전자선조사를이용한전처리는물리적방법이며장점으

로는

⁽¹⁾

^{, (2)}

결정화도의감소

^{, (3)}

^{, (4)}

^{, (5)}

^[1].

^,

^[15].

본연구에서는우리나라에서양이매우풍부한볏짚을대

상으로볏짚의전자선조사및

^NMMO

^,

처리를수행하였다

^{. NMMO}

X-

^FT-IR

의물리화학적변화를분석하여이를바탕으로새로운전처 리방법으로서의가능성을검토하였다

^.

재료 및 방법

실험재료

본연구에사용된볏짚은한국원자력연구원첨단방사선연 구소의육종시험농장에서

²⁰¹³

500

^m

⁶⁰

^C

12

. NMMO(50% in water, 4.8 mol/l)

TCI

Celluclast 1.5L (Cellulase 1.5L, Novo Co., Denmark)

Novozyme-188 (

-glucosidase, Novo Co., Denmark)

(Sigma-Aldrich Co., USA)

(sodium citrate buffer, 0.1 M, pH 4.8)

^.

전처리 방법

전자선조사를위하여건조된볏짚을

cell culture

polystyrene bottle

^{3 mm}

^.

^{2.5 MeV}

(ELV-8 type, EB Tech. Co., Korea)

^{25 mA}

^{, 25 kGy/}

scan

^,

^{10 m/min}

^{100, 300,} 500 kGy

^.

^{15 g}

^NMMO

^{285 g}

⁽

^{: 5 wt%)}

^NMMO

^{10 bar}

¹²⁰

^C

¹

^Whatman

No.1

과및세척한후잔류수분의제거를위하여

⁶⁰

^C

¹²

^.

E100N, E300N, E500N

^.

효소 당화

전자선조사및

^NMMO

볏짚은 미국 신재생에너지연구소

(National Renewable Energy Laboratory, NREL)

[24]. 5% (w/v)

(0.1 M, pH 4.8)

Celluclast 1.5L (70 FPU/ml)

^Novozyme- 188 (40 CbU/ml)

^(Jeio-Tech Co., Korea)

⁵⁰

^C

^{150 rpm}

24, 48, 72

^.

당수율 분석

전처리의효율을판단할수있는당수율분석을위하여

24, 48, 72

하여

^0.2

^m

^high

performance liquid chromatography (HPLC, Shimazu

Co., Japan)

^{. HPLC}

Aminex HPX-

87P column(Bio-Rad, USA)

RI detector

(410 refractive index detector, Waters, USA)

^{. HPLC}

^column

^detector

65

C

^column

(mobile phase)

수 증류수

(B&J HPLC certified solvent, SK chemical, Korea)

0.6 ml/min

^.

^.

성분 분석

전처리조건에따른볏짚의성분변화와당수율의분석은

NREL

NREL Laboratory Analytical Procedures [22, 26]

^TAPPI

^(TAPPI standards and suggested methods) [29]

^.

ATR-FTIR

전처리조건에따른볏짚의화학구조의변화를분석하기

위하여적외선분광기의전반사측정법

(ATR-FTIR Bruker

Vertex 70 spectrometer, Germany)

^{4 cm}

64

4,000-500 cm

^.

X-ray Diffraction

X-

(crystallinity

index, CrI)

부분이얼마나제거가되었는지판단할수있는기준이라고 할수있다

^.

^.

^(CrI)

^X-

(X'pert Powder, PANalytical, Netherlands)

40 kV, 30 mA, Cu K

⁽

^{= 1.54}

^),

¹

^{/min, 0.03}

^2θ

^5-50

^.

^Segal

^[23]

^. I

^{2θ = 22.4}

I

^{2θ = 18.4}

^.

Crystallinity Index (CrI,%) =

결과 및 고찰

볏짚의 성분변화

볏짚의구성성분과전처리조건에따른구성성분의상대 적인함량변화를

^{Table 1}

^.

^41.1%

^25.3%

66.4%

(holocellulose),

^19.8%,

질

^13.8%

^.

결과전자선

^{500 kGy}

함량변화는미미하였으나

^NMMO

66.4%

^74%

^7.6%

^{500 kGy}

사후

^NMMO

소의상대적인함량은전처리전의

^66.4%

^84.2%

17.8%

^.

리그닌과이물질이제거됨으로인하여상대적인함량이증 가한것이다

^.

^,

^{[2, 3].}

력하에고온에서

^NMMO

제거하였다

^.

^{, NMMO}

^NMMO

으로판단된다

^[31].

의상대적인함량감소와동시에전섬유소의상대적인함량 의증가로인하여효소당화시효소접근성과동시에당수율 을높일것으로예상할수있다

^.

당수율 분석

기존의연구결과들은리그노셀룰로오스별이론적당량

(

^,

⁾

일반적이다

^{. Table 2}

⁷²

^44.39%

^32.57%

^.

^,

선 단일 전처리 시

^{500 kGy}

58.6%, NMMO

^72%

^.

^NMMO

36.9%, 45.7%

5.8%, 13.8%

^.

사후

^NMMO

당수율

^(%)=

^(g)

¹⁰⁰

^(g)

I_cry–I_am Icry

--- 100×

Table 1. The compositional changes of the rice straw using different pretreatment methods.

Samples Holocellulose % Lignin % Ash %

Untreated 66.4 ± 0.2 19.8 ± 0.3 13.8 ± 0.5

E500 66.4 ± 0.3 19.8 ± 0.3 13.8 ± 0.6

NMMO 74.0 ± 0.3 18.1 ± 0.3 7.9 ± 0.4

E100N 79.8 ± 0.7 15.1 ± 0.4 5.1 ± 0.6

E300N 81.9 ± 0.6 13.2 ± 0.5 4.9 ± 0.5

E500N 84.2 ± 0.8 11.3 ± 0.7 4.5 ± 0.3

코스의경우전자선조사량에비례하여증가하는경향을보 였고

100, 300, 500 kGy

51.9%, 56.6%, 60.8%

^.

^,

^NMMO

^NMMO

^13.8%

11.8%, 11.1%, 9.7%

^.

^{Table 1}

^.

^,

^,

^.

^70-80%

(furfural),

(HMF)

소당화를저해한다고알려져있다

^[12].

생성물이발생하지않으며전처리에사용된

^NMMO

율은약

^98%

^NMMO

각

^{43%, 15%}

^{. Fig. 1}

^.

(41.3-44.3%)

^{24, 48,} 72

E100N (70.0%, 75.9%, 79.2%), E300N (72.1%, 77.9%, 81.8%), E500N (77.2%, 79.5%, 83.8%)

^,

²⁴

^70%,

72.1%, 77.2%

처리의효율을입증하였다

^.

적외선 분광 분석

적외선분광분석결과주요피크들은

^{Fig. 2}

와같이

^{3,420 cm}

^{, 2,919 cm}

1,300-1,800 cm

^{. 3,420 cm}

^-OH

[20]. 2,919 cm

Table 2. Sugar yields (%) of untreated and pretreated rice straw after 72 h of enzymatic hydrolysis.

Samples Glucose Xylose Mannose Galactose Arabinose Total

Untreated 32.57 ± 0.6 - 10.64 ± 0.3 0.35 ± 0.1 0.83 ± 0.1 44.39 ± 0.5

E500 36.94 ± 0.7 5.85 ± 0.7 12.93 ± 0.2 0.98 ± 0.1 1.91 ± 0.2 58.63 ± 0.6

NMMO 45.70 ± 0.5 13.89 ± 0.8 10.45 ± 0.3 0.25 ± 0.1 2.36 ± 0.2 72.65 ± 0.4

E100N 51.96 ± 0.6 11.89 ± 0.7 12.38 ± 0.3 0.25 ± 0.1 2.72 ± 0.2 79.20 ± 0.6

E300N 56.69 ± 0.8 11.10 ± 0.6 11.02 ± 0.2 0.28 ± 0.1 2.73 ± 0.1 81.82 ± 0.5

E500N 60.83 ± 0.7 9.74 ± 0.9 10.85 ± 0.4 0.28 ± 0.1 2.19 ± 0.2 83.89 ± 0.5

Fig. 1. Variations in the sugar yields based on the enzymatic hydrolysis time (24, 48, and 72 h) in untreated and pretreated samples.

Fig. 2. FT-IR Spectra in the range of 4,000-500 cm

indicating

changes in the chemical structures of untreated and E500N

sample.

의피크는셀룰로오스의메틸렌구조중

^C-H

[16]. 1,731 cm

(ester linkage)

^.

^{1,596 cm}

1,624 cm

^-

^[4].

897 cm

^-1,4-

⁽

-1,4-glucosidic

bond)

이는셀룰로오스의상대적함량이증가한것을의미한다

^.

X-선 회절 분석

Table 3

의효율또한증가한다

^.

43.5%

^E500N

^56.3%

^12.8%

^.

^.

요 약

2

^NMMO

^.

500 kGy

^NMMO

⁷²

^60.8%

^9.7%

^83.8%

정도의당수율을보였다

^.

^NMMO

^.

사량이너무높다는단점이있으며추후에는낮은전자선조 사량과여러가지단일공정을통합한새로운복합전처리방 법연구의필요성이있다

^.

자선조사라는친환경적방법과더불어

^NMMO

활용성을이유로바이오에탄올생산등의산업현장에의적 용가능성이높다는것을알수있었다

^.

Acknowledgments

This study was supported by the Nuclear R&D program of the Korea Science and Engineering Foundation, which is funded by the Ministry of Science, ICT and Future Planning of the Republic of Korea.

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