Journal of Materials Science and Engineering B 3 (10) (2013) 670-676

D

DAVID

PUBLISHING

Research to Produce Ethanol from Seaweed Biomass Cladophora sp Vo Thanh Trung1, Bui Minh Ly1, Le Nhu Hau1 and Nguyen Thanh Hang2 1. Nhatrang Institute of Technology Reasearch and Application, Nha Trang, Viet Nam 2. Hà Nội University of Science and Technology, Ha Noi, Viet Nam Received: April 28, 2013 / Accepted: June 01, 2013 / Published: October 25, 2013. Abstract: Biomass seaweed Cladophora sp. (BSC) included Cladphora socialis, Cladophora prolifera, Cladophora crupila, which was hydrolysised in dilute sulfuric acid and enzymatic hydrolysis of cellulase. BSC was treated by dilute sulfuric acid at concentration of 0, 0.5%, 1%, 2%, 3%, 4%, 5%, 6% (v/v) (v: volume) in autoclave respectively at 121 °C at time 20, 40 and 60 minute, with the rate between seaweed and solution acid of 1/10(w/v) (w: weight). Beside, BSC was pretreated in dilute sulfuric acid at 0.5%(v/v) at 121 °C in 20 minute, after it was treat at concentrations 0, 0.2, 0.4, 0.6, 0.8, 1 ml/g with the enzymatic hydrolysis consisted of cellulase in control (50 °C, pH 5.0, 24 h). The results showed that, total sugar contents of two methods are the same, but sugar content was varied by treatment time of heat. Rates of acid 4%; 5%, 6% (v/v) and 0.8; 1 ml/g have high sugar content within the range of 42.38-48.51 mg/ml. 400 ml of hydrolysis solution was fermented by Saccharomyces cerevisiae at 30 °C for 72 h. After fermentation, 5% ethanol solution was identified for the first 100 ml evaporate solution. BSC is easily hydrolyzed and fermented to ethanol, this shows excellent prospects as a potential feedstock for the production of bioethanol. Key words: BSC, Cladophora sp, green seaweed biomass, ethanol green seaweed, carbohydrate seaweed.

1. Introduction Seaweeds have large biomass and high total carbohydrate. Seaweed should be choice suitable material for ethanol research. In the present, many countries are focusing on ethanol production from raw seaweed, but this material resources of each country is different, due to depends on the characteristics of the distribution of species and the technical situation of each country. Production of high concentrations of bioethanol from seaweeds, that has contains polysaccharides easily hydrolyzable. Three type polysaccharides of seaweed, sea lettuce, chigaiso, and agar weed, were used as representatives of green, brown, and red seaweeds, respectively, and methods for obtaining high concentrations of ethanol (bioethanol) from these Corresponding author: Vo Thanh Trung, research fields: seaweeds biomass, biofuel. E-mail: [email protected].

seaweeds were investigated. Starch, B -1,3-glucan, and cellulose were present as glucans in the seaweeds examined, though the cellulose content was relatively low compared to that in lignocellulose However, little to no lignin was present in the seaweeds to interfere with the hydrolysis of polysaccharides, making the polysaccharides in these seaweeds easily hydrolyzable [1]. The marine ecosystem has vast resources of biomass with high to very high carbohydrate percentage. The marine biomass macro algae having very good potential for bioethanol production marine algae Enteromorpha species is used as the starting material for the bioethanol production in the present investigation. This algae is very rich in carbohydrate source ranging from 70%-72%. Successful bioconversions of algal biomass to ethanol have been achieved by a series of different pretreatment, hydrolysis and fermentation [2]. The following to Ref. [3], “Brown seaweed contains

671

Research to Produce Ethanol from Seaweed Biomass Cladophora sp

biomass, short growing cycle (21 days), harvest yield dry 22 tons/ha/year. Enzyme Visocozyme L is collected from Aspergillus sp. chains. This enzyme has to have an effect to groud polysaccharids (arabinose, celluose, B-glucan, hemicellulose, xylan), active (1000 UI dry). This enzyme purchased from Sigma Chemical Co., USA. Enzyme Visocozyme L was diluted of 20 time rate before hydrolysis. Yeast (Saccharomyces cerevisiae) is name THERMOSACC® DRY imports by Nam Giang Co., Ltd. Viet Nam.

various carbohydrates, such as alginate, laminaran, and mannitol, therefore ethanol fermentation was attempted with Nuruk and a mixed culture that included Laminaria japonica. Nuruk is used to make Korean traditional alcohol”. Laminaria digitata is a highly prevalent kelp growing off the coast of the UK but has rarely been considered as a source of biomass to date. It can be used as a feedstock in both ethanol fermentation and anaerobic digestion for methane production [4]. Hydrolysis of invasive Algal Feedstock for Ethanol Fermentation was to develop a saccharification method for the production of third generation biofuel (i.e., bioethanol) using feedstock of the invasive marine macroalga Gracilaria salicornia. Batch fermentation using the ethanologenic strain Escherichia coli KO11. Furthermore, ethanol production kinetics indicated that, the invasive algal feedstock contained different types of sugar, including C5-sugar. This study shows that, there is great potential for the production of renewable energy using marine invasive biomass [5]. The following to (Leilei Ge et al., 2011) [6], “Floating residue (FR), a surplus by-product from the alginate extraction process, contains large amount of cellulosic materials. FR is easily hydrolyzed to produce glucose in comparison with that in terrestrial plants. FR shows excellent prospects as a potential feedstock for the production of bioethanol”. In Vietnam, though the survey process of biological characteristics and seaweeds resources. We decided to select seaweed Cladophora sp. for ethanol research.

This method is sometimes necessary to quantify the

2. Materials and Methods

amount of sugar in a certain medium. Whether the

2.1 Raw Material and Yeasts

residues, or attached to a polymer, the phenol-sulfuric

Cladophora sp. was collected at latiue of VietNam. Cladophora sp. is a green seaweeds population, which include Cladophora socialis, Cladophora cripula, Cladophora prolifera species [7]. They have big size and biomass. BSC does not affect food security and uncompetitive agricultural land. BSC is absorption of CO2 and nutrients to improve the environment. BSC is ability to grow and develop well to create large

2.2 Chemical Analysis 2.2.1 Chemical Composition Analysis Total nitrogen, fiber, and ash contents were determined by standard AOAC (1990) [8] methods. Fat content was determined according to Bligh and Dyer method [9]. Total protein content was calculated by multiplying Kjeldahl nitrogen by 6.25. Ash content was conducted by ashing the ground dried samples overnight in muffle furnace at 525 °C. Total carbohydrate is calculated by difference (= 100-crude protein-crude lipid-ash-huimidity) [10]. Cellulose was isolated from the seaweeds following the method described by Ref. [11]. 2.2.2 Total Sugar Content of Hydrolysis Solution Analysis Total sugar was estimated by Dubois method [12].

sugar is in the presence of various salts or protein acid assay can be performed. Determination of sugars using phenol-sulfuric acid is based on the absorbance at 490 nm of a colored aromatic complex formed between phenol and the carbohydrate. 2.2.3 Alcohol Analysis Hydrolysis

solution

was

fermented

by

Saccharomyces cerevisiae. This solution is evaporated, before determination ethanol volume. Ethanol volume is

672

Research to Produce Ethanol from Seaweed Biomass Cladophora sp

measured by boiling point determination method [13].

Dry BSC

2.3 Dilute acid and Enzymatic Hydrolysis 2.3.1 Pretreatment Raw Material The fresh BSC is dried in the sun until dry seaweed after harvest. Then put BSC in fresh water and stir to wash the salt and reduce the amount of impurities sticking on BCS. After washing fresh, BSC seaweed was picked and dried sun in the second until the moisture content of 5%-10% BCS. Then BSC is be milled to small, which will be packeted to prepare for hydrolysis. 2.3.2 Dilute acid Hydrolysis The acid hydrolysis is described in Fig. 1. The dilute acid treatment of BSC was optimized at 121 oC in an autoclave for different sulfuric acid concentrations (0, 0.5%, 1%, 2%, 3%, 4%, 5%, 6% v/v) and different time (20, 40 and 60 min). After treatment, solution sugar is clean filtrated impurity and then analyzed for total reducing sugar. The rate between BSC and solution acid of 1/10 (w/v), the same each experiment have is 5 g dry weight BSC hydrolyzed in 50 ml of H2SO4. A mixture of seaweed and acid is contained in 100 ml bottle and a lid. The experiments were performed in triplicate. 2.3.3 Enzymatic Hydrolysis The enzyme hydrolysis is described in Fig. 2. BSC was pretreated in dilute sulfuric acid at 0.5%(v/v) at 121 °C in 20 min, after it was treat at concentrations 0, 0.2, 0.4, 0.6, 0.8, 1 ml/g with the enzymatic hydrolysis consisted of cellulase in control (45 °C; pH = 5.0; in time 12, 24, 30 h). The hydrolysis was performed in a laboratory incubator at 150 rpm and 45 °C for 30 h. The experiments were performed in triplicate.

Conentration acid

0%

0.5

1%

2%

3%

6%

5%

4%

Sugar content analysis Fig. 1 Dilute acid hydrolysis diagram. Dry BSC

Pretreatment with dilute acid H2SO4 0.5 % C (v/v)

Enzymatic hydrolysis

0 ml

0.2

0.4

0.6

0.8

1 ml

Total sugar analysis Fig. 2 Enzymatic hydrolysis diagram.

solution. Then this solution was fermented with dry yeast (Saccharomyces sereveace) at a temperature of 30 °C in 72 h. The product of this process is ferment solution. Then this solution is evaporated and determining the volume alcohol.

3. Results

2.4. Ethanol Fermentation

3.1 Chemical Composition Raw Materials

After hydrolysis, the sugar solution will be purify and fermented ethanol with dry yeast. The fermentation process is described in Fig. 3. Sugar solution of hydrolysis was purified, neutralized to pH 5.0 and determine the total sugar content of the

The proximate composition based on dry weight of Cladphora socialis, Cladophora prolifera, Cladophora crupila were shown in Table 1. It was found that the protein, lipid, ash, humidity and carbohydrate content were evaluated to estimate of tree species. Cladophora

Research to Produce Ethanol from Seaweed Biomass Cladophora sp Sugar solution

Purify

Exclude waste

Adjust pH

Fermentation

Ferment solution

Evaporate

Ethanol Fig. 3 Ferment ethanol diagram.

seaweeds always content humidity from 10%-15 % because it has ability to absorb water. Ash content of seaweeds are not high (about 7%-10%). The carbohydrate content of tree species is the highest and the lipid content is the lowest. Carbohydrate of species about 58%-61%, which three times higher protein content ( about 15%-20%) and thirty times higher lipid (about 1-2.5), beside content cellulose about 51% dry weight of seaweeds. Therefore (BSC) is suitable for ethanol production rather than food production from sources protein, and production diesel fuel from lipid sources. Special the following to research result of (Isa et. al., 2009) [1], the carbohydrate content of Table 1

Cladophora prolifera have 78 glucose percent, 8 arbinose percent, 10 galactose percent and 4 xylose percent. Cladophora prolifera is a species of BSC so the content monosacchrids of BSC sepices are estimate. Glucose is a important monosacharid for ethanol fermenter, therefor BSC is suitable raw material for ethanol production. 3.2 Result of Dilute acid Hydrolysis Method

Sterilize Yeast (Saccharo myces sereveace)

The results form Table 2 and Fig. 4 show that BSC was hydrolyzed by high heat and different space time in dilute acid to make various content sugars. The result of table and image was found that content sugar solution has fluctuated to follow to hydolysis time. BSC was hydrolyzed in 20 min to created sugar content lower than 40 and 60 min. Sugar conten was created of hydrolysis 40 min and 60 min as the same. The content sugar of experiments was increasing fluctuated from low to high concentration. When the hydrolysis have not acid, the sugar content is the lowest 4.47 mg/ml but add 5% concentration is the highest sugar content 47.47 mg/ml. In the dilute acid hydrolysis, the 0%, 1%, 2% acid concentration have low efficiency, sugar content fluctuate between 4.74 and 19.45 (mg/ml). But 3, 4, 5, 6% acid concentration have hydrolysis high efficiency, the sugar content fluctuate between 29.57 and 47.47 mg/ml. Inside the sugar content are created by 3% concentration (24.57 mg/ml) lower than the levels 4%, 5% and 6%. Sugar content was created by a concentration of 4% = 41.31 (mg/ml), 5% = 47.47 (mg/ ml) and 6% = 47.24 (mg/ml) are approximately equivalent. This shows that, the limit in the process of

Chemical composition of Cladphora socialis, Cladophora flexuosa, Cladophora crupila.

Contents Humidity % Ash % Protein % Lipid % Carbohydrate % Cellulose%

Cladphora socialis 11.5 7.71 20.89 1.52 58.38 50.15

673

Cladophora prolifera 13.4 7.76 15.04 2.06 61.74 53.25

Cladophora crupila 13.1 8.4 17.35 2.37 58.78 51.42

Cladophora sp. (BSC) 12.67 7.96 17.76 1.98 59.63 51.61

Note: Cladophora sp. (BSC) contents = average contents (Cladphora socialis, Cladophora prolifera, Cladophora crupila)

674

Research to Produce Ethanol from Seaweed Biomass Cladophora sp

Table 2

Average and standar deviation total sugar content of experiments of hydrolysis acid method. C acid (%v/v)

Time

0 3.65 ± 0.11 5.36 ± 0.1 5.21 ± 0.07 4.74

20 m 40 m 60 m Average

1 7.24 ± 0.1 12.22 ± 0.11 12.78 ± 0.11 10.75

The content sugar hydrolysis by acid (mg/ml) 2 3 4 17.21 ± 0.33 26.28 ± 0.38 40.31 ± 0.37 19.59 ± 0.11 30.76 ± 0.29 41.42 ± 0.4 21.55 ± 0.09 31.66 ± 0.41 42.19 ± 0.17 19.45 29.57 41.31

Su g ar so lu tio n (m g /m l)

60 50 40 30 20 10 0 0

1

2 20 m

3 40 m

4

5 60 m

6

C acid (%v/v)

Fig. 4 Fluctuations sugar content follow to concentration of acid in acid hydrolysis.

hydrolysis, only add 5% acid concentration is hydrolyzed thoroughly. 3.3 Result of Hydrolysis Enzyme Enzymatic hydrolysis method, BSC is pretreated in 0.5% acid conentration medium and high heat, to create conditions for enzyme in direct contact with the substrate to the reaction easily. The results Table 3 and Fig. 5 show the sugar content is created from this method is quite high, fluctuate between 5.35 and 39.67 mg/ml. The sugar content was created after 12 h of hydrolysis is lower than 24-30 h of hydrolysis at all concentration levels of the enzyme. The sugar contents are fluctuated to follow concentration levels of different enzymes. The enzyme concentrations of 0, 0.2, 0.4 ml were Table 3

12 h 24 h 30 h Average

6 46.01 ± 0.2 47.53 ± 0.34 48.19 ± 0.24 47.24

created low sugar content fluctuate between 5.35 and 17.88 mg/ml, but the enzyme concentrations of 0.6, 0.8, 1 ml were created high sugar content fluctuate between 27.59 and 39.67 mg/ml. This indicates that, while increasing the concentration of enzyme hydrolysis produces the sugar content increases, but continue to increase the amount of enzyme to 1 ml, the sugar content did not increase. Thus the amount of enzyme was 0.8 ml was sufficient for hydrolysis takes place thoroughly, if you add too much enzyme then the excess and waste enzymatic. Sugar content is created by enzyme hydrolysis method equal nearly compared with dilute acid hydrolysis method. Sugar content is created of enzyme hydrolysis method is 42.38-43.42 mg/ml and hydrolyzed acid method is 46.31-47.47 mg/ml.

3.4 Hydrolysis Efficiency and Fermentation The results Table 4 show that there is always a loss of high sugar in the process of hydrolysis. Sugar recover content is often accounted for 41% to 43% of weight BSC hydrolysis. It is equivalent to a recover solution about 90 to 96% of hydrolyzate solution. Solution after hydrolysis is neutralized to pH = 5 then it is fermented with Saccharomyces serevisiae. A 400 ml volume of sugar solution has 45 mg/ml concentration equivalent to sugar content of ferment process is 18 g, Yeast content is used 0.2 g. Ferment

Average and standar deviation total sugar content of experiments of hydrolysis enzyme method.

C enzyme (ml/g) Time

5 46.34 ± 0.39 48.51 ± 0.2 47.55 ± 0.21 47.47

0 5.3 ± 0.17 5.51 ± 0.37 5.24 ± 0.21 5.35

The content sugar hydrolysis by enzyme (mg/ml) 0.2 0.4 0.6 0.8 8.38 ± 0.23 14.73 ± 0.24 23.17 ± 0.26 30.47 ± 0.35 12.46 ± 0.28 18.51 ± 0.23 29.11 ± 0.27 42.38 ± 0.39 13.55 ± 0.32 20.41 ± 0.29 30.49 ± 0.17 43.21 ± 0.23 11.46 17.88 27.59 38.69

1 32.19 ± 0.33 43.42 ± 0.34 43.4 ± 0.24 39.67

Research to Produce Ethanol from Seaweed Biomass Cladophora sp

S u g a r s o lu t io n ( m g /m l)

50 45 40 35 30 25 20 15 10 5 0 0

0.2

0.4 12h

0.6 24h

0.8 30h

1 C enzyme (ml/g)

Fig. 5 Fluctuations sugar content follow to concentration of enzyme in enzyme hydrolysis. Table 4 Hydrolysis efficiency and fermentation. Component Value Solution before hydrolysis 50(ml) Solution after hydrolysis 45-48(ml) The recover efficiency of the hydrolysis 90-96% solution Weight BSC hydrolysed 5 (g) Total sugar analysis (mean of experiments 45.65 (mg/ml) have high sugar content) Weight sugar recover 2.054-2.191 (g) Hydrolysis efficiency 41.08%-43.82% Volume of ferment solution 400 (ml) Sugar content fermented 45 (mg/ml) Yeast (Saccharomyces cerevisiae) 0.2 (g) Volume alcohol after evaporate 5% Hydrolysis efficiency = Weight sugar recover × 100/ Weight BSC hydrolysed

process happen at 30 °C in 72 h control. After fermentation, 5% ethanol solution was identified for the first 100 ml evaporate solution. BSC is green seaweeds, total Carbohydrate have main cellulose content, therefore hydrolysis process of this biomass have efficiency is lower than red and brown seaweeds.

4. Discussion BSC hydrolysis by acids and enzymes are two completely different hydrolysis methods. Hydrolysis enzyme method has the highest sugar content, when BSC is hydrolyzed in conditions (enzyme concentration of 1 ml/g, pH 5.0, 24 h). Hydrolysis dilute acid method has the highest sugar

675

content, when BSC is hydrolyzed in conditions (acid concentration of 5%, temperature 121 °C in 40 min). Sugar content is created the highest of hydrolysis enzyme method equal nearly compared with hydrolysis dilute acid method. Sugar content is created of enzyme hydrolysis method is 42.3-43.4 mg/ml and hydrolyzed acid method is 41 to 47 mg/ml. BSC is green seaweeds, total Carbohydrate have main cellulose content, therefore hydrolysis process of this biomass have efficiency is lower than red and brown seaweeds. Hydrolysis efficiency from BSC is 41%-43% lower than the hydrolytic performance from red and brown seaweed from 10%-15%. According to the authors [14] hydrolysis performance from red seaweed Echeuma spp. 0.7 kg/kg, respectively 70%, and according to the authors [15] performance from the brown seaweed Laminaria spp hydrolysis is 55%. The difference can be explained because the total Carbohydate content of red and brown seaweed more green 5-10%, in addition to the main components Carbohydrate of Red seaweed is Carragenan or Agar, brown seaweed alginate and fucoidan [16] more to hydrolyze. In the opposite, ethanol was fermented from red and brown seaweed more difficult than fermented from green seaweeds, because hydrolysis solution of green seaweed has contain more sugar glucose than red and brown seaweed.

5. Conclusions Total sugar content of two methods is the same, but sugar content was varied by treatment time of heat. Concentration of acid 5%, 6% (v/v) and concentration of enzyme 0.8; 1 ml/g were hydrolysis to create high sugar content within the range of 42.38-48.51 mg/ml. a volume 400 ml of hydrolysis solution was fermented by Saccharomyces cerevisiae at 30 °C in 72 h. After fermentation, 5% ethanol solution was identified for the first 100 ml evaporate solution. BSC is easily hydrolyzed and fermented to ethanol, it shows excellent prospects as a potential feedstock for

676

Research to Produce Ethanol from Seaweed Biomass Cladophora sp

the production of bioethanol.

Acknowledgments I would like to thank Vietnam Academy of Science and Technology and Hà Nội University of Science and Technology has created favorable conditions for us to perform this study.

References [1]

[2]

[3]

[4]

[5]

[6]

[7] [8]

M. Yanagisawa, K. Nakamura, O. Arigab, K. Nakasaki, Production of high concentrations of bioethanol from seaweeds that contain easily hydrolyzable polysaccharides, Process Biochemistry 46 (2011) 2111-2116. S. Nahak, G. Nahak, R.K. Sahu, Bioethanol from marine algae: A solution to global warming problem, Journal of Applied Environmental and Biological Sciences (2011) 74-80. S.M. Lee, J.H. Lee, The isolation and characterization of simultaneous saccharification and fermentation microorganisms for Laminaria japonica utilization, Bioresource Technology 102 (2011) 5962-5967. J.M.M. Adams, T.A. Toop, I.S. Donnison, J.A. Gallagher. Seasonal variation in Laminaria digitata and its impact on biochemical conversion routes to biofuels, Bioresource Technology 102 (2011) 9976-9984. X. Wang, X. Liu, G. Wang, Two-stage hydrolysis of invasive algal feedstock for ethanol fermentation, Journal of Integrative Plant Biology 53 (3) (2011) 246-252. L. Ge, P. Wang, H. Mou, Study on saccharification techniques of seaweed wastes for the transformation of ethanol, Renewable Energy 36 (2011) 84-89. P.H. Hộ, Seaweeds in Việt Nam, Science Publishers Vietnam,1969, p. 558. Official Methods of Analysis, 16th ed., Association of

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

Official Analytical Chemists, Washington D.C., AOAC, 1990. E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification Can. J. Biochem. Physiol 37 (1959) 911-917. P. Márcia, S.G.F. Paulo, L.M. Alvaro, Chemical composition of ulvaria oxysperma, ulva lactuca and ulva fascit, Brazilian Archives of Biology and Technology 47 (1) (2004) 49-55. A.K. Siddhanta, K. Prasad, R. Meena, G. Prasad, G.K. Mehta, M.U. Chhatbar, et al., Profiling of cellulose content in Indian seaweed species, Bioresour. Technol. 100 (2009) 6669-6673. J. Wiley, Determination of reducing and nonreducing sugars using the phenol-sulfuric acid assay, Current Protocols in Food Analytical Chemistry (2001) E1.1.1-E1.1.8 T. Minna, L. Pekka, Determination of the alcoholic strength of grape and fruit wines by simple analytical methods Am, J. Enol. Vitic. 48 (1997) 220-224. C.S. Goh, K.T. Lee, A visionary and conceptual macroalgae-based third-generation bioethanol (TGB) biorefinery in Sabah, Sustainable Energy Reviews (2010) 842-848. J.M. Adams, I.S. Donnison, Fermentation study on Saccharina latissima for bioethanol production considering variable pre-treatments, J. Appl. Phycol. 21 (2009) 569-574. G.S. Kim, M.K. Shin, Y.J. Kim, K.K. Oh, J.S. Kim, H.J. Ryu, et al., Method of producing biofuel using sea algae, International application published under the patent cooperation treaty (PCT), WO2008/105618, 2008. A. Isa, Y. Mishima, O. Takimura, T. Miniwa, Preliminary study on ethanol production by using macro green algae, Journal of the Japan Institute of Energy 88 (2009) 912-917.