Bioresource Technology 135 (2013) 150–156

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Biore source Tec hnology

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Bioethanol production from Gracilaria verrucosa , a red alga, in abiorefinery approach

0960-8524/$ - see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.10.120

⇑ Corresponding authors. Tel.: +91 9871509870; fax: +91 11 24115270. E-mail addresses: kuhad85@gmail.com (R.C. Kuhad), dbsahoo@hotmail.com

(D.B. Sahoo).

Savindra Kumar a, Rishi Gupta b, Gaurav Kumar a, Dinabandhu Sahoo a,⇑, Ramesh Chander Kuhad b,⇑a Marine Biotechnology Laboratory, Department of Botany, University of Delhi, Delhi 110007, India b Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India

h i g h l i g h t s

" First time use of algal pulp for bioethanol produc tion. " Pro duction of agar and ethanol in a biorefinery approach. " The ethanol yield obtained was comparatively higher than earlier report. " An integrated biorefinery approach has been discussed.

a r t i c l e i n f o

Article history: Available online 2 November 2012

Keywords:BiorefineryEthanolGracilaria verrucosa Algal pulp Mass balance

a b s t r a c t

In this study, Gracilaria verrucosa , red seawee d has been used for production of agar and bioethanol. The algae harvested at various time durations resulted in extraction of �27–33% agar. The leftover pulp was found to contain �62–68% holocellulose, which on enzymatic hydrolysis yielded 0.87 g sugars/g cellu- lose. The enzymatic hydrolysate on ferme ntation with Saccharom yces cerevisiae produced ethanol with an ethanol yield of 0.43 g/g sugars. The mass balance evaluation of the comp lete process demonstrates that developing biorefinery approach for exploiting Gracilaria verrucosa , a red alga, could be commercially viable.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction Olsen, 2011 ). These problems force to draw our attention toward

The increase in fossil fuel prices, issues of national security and environmental concerns have led to an overwhelmi ng interest among researche rs to develop economical ly viable process for pro- duction of alternativ e transportation fuels. Among these, bioetha- nol, a renewable source of energy, has been accepted most widely as the alternative to fossil fuels. Bioethanol market grew from less than a billion liters in 1975 to more than 86 billion liters in 2010 and is expected to reach 100 billion liters by 2015 (Licht,2006). Till date the commercial production of bioethan ol is being carried out from first generation substrate s, which will cause ‘‘Food verses Fuel’’ competition. Also, the excessive use of harmful pesti- cide and fertilizer in production of first generation crop plants also damage the environm ent (Kumar and Sahoo, 2012 ). Because of such issues, the production of bioethan ol from plant residues is opted, but the ethanol production from these feedstock s also has many hurdles such as high cost of production, structural character- istics, feedstock collection network and limited yield (Singh and

seaweeds , a diverse group of autotrophic organisms, as an alterna- tive substrate for the bioethanol production.

Like plants, seaweeds (marine algal feedstocks) are also com- prised of rigid cellulose-bas ed cell walls and accumulate various complex polysacchar ides, which can be hydrolyzed to sugars and subsequent ly be fermented to ethanol (Goh and Lee, 2010; Adams et al., 2011 ). Moreover, algal feedstock s have several advantages, which include high area productivity , no competition with conven- tional agriculture for land, utilization of different water sources (e.g., seawater, brackish water and wastewater), recycling of car- bon dioxide, and compatibility with integrated production of fuels and co-products within biorefineries (Sahoo et al., 2012 ). The use of marine biomass as a source of energy was investiga ted in US and Japan in 1970s after the oil crisis, but the studies were discon- tinued when oil prices stabilised (Yokoyama et al., 2007 ). In recent years there are several reports on ethanol production from various seaweeds (Adams et al., 2011, 2012; Khambhaty et al., 2012; Kim et al., 2011; Park et al., 2012 ), however , this area needs further interventi ons and innovations.

The seaweeds are known to contain other valuable materials in addition to the fermentabl e carbohydrat es, thereby providing carbohyd rates as byproducts after extractio n of economicall y

S. Kumar et al. / Bioresource Technology 135 (2013) 150–156 151

important products such as agar–agar and phycocol loids, etc. The utilization of no-value byproduct for ethanol production will econ- omize the bioprocess and eventually will meet the concept of developing biorefinery with zero-waste.

Here, we have attempted to use an alga Gracilaria verrucosa , the third largest genus of class Rhodophyta , for the production of agar and bioethanol as a biorefinery approach. To the best of our knowl- edge this is the first report on the production of bioethanol using pulp or algal byproduct after agar extraction. An efficient strategy for agar extraction from seaweed was developed and the resultant pulp was subsequently used for bioethanol production. Moreover, an attempt has been made to present a conceptual design of a bior- efinery system with an estimate d mass balance of the process.

2. Methods

2.1. Biomass collection and preparation

Field trips were undertaken to different parts of the Indian coast over a period of two years. Samples of G. verrucosa were collected from different sites of Chilika Lake, Odisha (19�280–19�540 N and 85�050–85�380 E) during the months of January, May and July 2009 as well as January, May and July 2010. The collected algal bio- mass was washed in tap water to remove sand and excess salt and dried at room temperature for 24 h. The dried algal biomass was cut into small pieces, sieved to attain a particle size of 0.5–1.5 mm and stored in sealed plastic bags for further processin g.

2.2. Agar extraction

The agar extractio n from G. verrucosa was carried out using al- kali treatment method (Istini et al., 1994 ). The processed oven dried biomass (50.0 g) was treated with 1.0 L of 5.0% NaOH solu- tion at 80 �C for 2 h. Thereafter, the biomass was washed in tap water and neutralized with 1.5% H2SO4 for 2 h. The treated biomass was boiled for 90 min with 1.0 L of distilled water and the filtrateobtained was gelled at room temperature. The solidified agar was then cut into small strips and kept for freezing at �35 �C for 24 h. The frozen agar was thawed in tap water, soaked in acetone and dried at room temperature . Remaining pulp was collected and dried at 60 �C for further analysis.

2.3. Characterizati on of residual pulp

The agar pulp was analyzed for the available total sugar, total reducing sugar content and glucose following modified TAPPI(1992) protocols. For this, 0.3 g of biomass was treated with 3 mL of 72% H2SO4 (v/v) in a 25 mL beaker with magnetic stirrer bar at 25 �C for 30 min. The reaction mixture was then diluted with dis- tilled water to maintain a final acid concentr ation of 2.5%. The sus- pension was then hydrolyzed in an autoclave (30 min, 121 �C,1 bar). The hydrolyzed material was cooled down, neutralized with NaOH to determine reducing sugar by Dinitrosalicy lic Acid (DNSA)reagent (Miller, 1959 ), Glucose by GOD–POD kit (Span Diagnost ics Ltd., India) and total carbohydrat e estimation according to Duboiset al. (1956).

2.4. Enzymatic hydrolysis of algal pulp

Commercial cellulase from Trichoderma reesei (ATCC 26921) and b-glucosid ase from Aspergillus niger (Novozyme 188) were pur- chased from Sigma (St. Louis, MO, USA).

The residual pulp of G. verrucosa was suspended in 0.05 M citrate phosphate buffer (pH 5.0) at 50 �C with solid content of 10% (w/v)and soaked in rotatory incubator shaker (Innova 4400, New

Brunswic k Scientific, NJ, USA) for 2 h. Then, the suspension was supplem ented with cellulase (20 FPU/g dry substrate) and b-glucosidas e (60 U/g dry substrate ) and the reaction continued at 50 �C and 150 rpm. Each reaction mixture was supplemented with 0.005% (w/v) sodium azide to avoid microbial contaminat ion and 1.0% (v/v) Tween 80 to facilitate the enzymatic action. The samples were withdraw n every 6 h and subsequently analyzed for the glucose released in the reaction mixture using DNSA reagent (Miller, 1959 ).

2.5. Ethanol fermentation

Saccharom yces cerevisiae HAU strain was maintained on YPD- agar plates containing (g/L); glucose 30, yeast extract 3.0, peptone 5.0, agar 20.0 at pH 6.0 ± 0.2 and temperature 30 �C The inoculum was develope d by growing the yeast cells in the YPD broth to an OD of �0.6 as described elsewhere (Gupta et al., 2009; Kuhad et al., 2010a ).

The fermentation of enzymatic hydrolysate s was carried out separately in 250 mL Erlenmeyer flasks with working volume of 50 mL. The enzymatic hydrolysate supplemented with 3 g/L yeast extract and 0.25 g/L (NH4)2HPO4 was inoculated with S. cerevisiae (6.0% v/v) at pH 6.0 ± 0.2. Samples were withdrawn at 6 h intervals and centrifuged at 10,000 �g for 15 min at 4 �C. The cell free super- natant was evaluated for ethanol and residual sugar concentration.

2.6. Analytica l methods

The chemical composition analysis of algal pulp for holocellu -lose, cellulose, hemicellulose, lignin, ash, moisture content was carried out using TAPPI (1992) protocols. The reducing sugars ob- tained after enzymatic saccharification were estimated according to Miller’s method (1959). The saccharification efficiency of the al- gal pulp was calculated as follows:

Saccharification efficiency ð%Þ¼ Amount of sugar released during the hydrolysis Amount of holocellulose present in the initial substrate �1:11

3. Results and discussion

Worldwid e Gracilaria species are not only used for agar produc- tion but also as feed for abalone and as a biofilter in aquaculture. G.verrucosa is known for its wider adaptability which can grow in both seawater and brackish water. Large scale cultivation of Graci-laria was initiated in Chilika Lake and other parts of India and the cultivatio n methods have been well standardi zed (Sahoo et al., 2003). It has been estimate d that more than six tons of G. verrucosa (fresh weight) can be produced per acre of water area.

3.1. Change in agar yield, carbohydrates and pulp content of G. verrucosa

Agar yield from G. verrucosa sp. was found to vary as a function of seasons and life stage (Table 1). The maximum agar was ex- tracted from the algal mass harvested in July 2010 (32.53%), fol- lowed by those in July 2009 (32.53 ± 1.42%). While, the algal samples collected in May 2009 were observed to give the lowest agar yield (26.83 ± 1.87%). Based on the agar yield (percentagedry weight) and the biomass (dry weight) profile of G. verrucosa from the fixed area, it was observed that for commercial extraction of agar the harvesting of G. verrucosa should be done in month of July. Agar concentratio n in dry weight reached a maximum of 32.53 ± 1.42% in July 2010 and a minimum of 26.83 ± 1.87% in May 2009. While, pulp content varied between 23.57 ± 0.93 and 26.27 ± 0.85% (dry weight basis) from January 2009 to July 2010. The total carbohydrat e concentr ation in G. verrucosa biomass

Table 1Seasonal change in agar and biochemical properties of G. verrucosa .

Time of sampling Agar (%) Pulp (%) Moisture (%) Salt and silt (%) Ash (%) Other components (%) Total carbohydrates (%)

January 2009 30.83 ± 1.05 23.57 ± 0.93 4.5 21 ± 1 13.6 ± 0.40 6.5 41.83 ± 1.04 May 2009 26.83 ± 1.87 24.17 ± 0.61 4.6 22.5 ± 0.5 13.07 ± 0.32 8.83 42.67 ± 1.15 July 2009 31.57 ± 0.81 25.33 ± 1.75 4.5 21 ± 0.5 12.73 ± 0.21 4.87 43.50 ± 1.32 January 2010 29.67 ± 2.06 24.50 ± 0.26 4.7 21 ± 1 13.97 ± 0.15 6.16 41.00 ± 1.0 May 2010 28.67 ± 0.29 24.80 ± 0.56 4.5 22.8 ± 0.52 13.37 ± 0.31 5.86 43.00 ± 1.0 July 2010 32.53 ± 1.42 26.27 ± 0.85 4.4 21 ± 0.5 12.73 ± 0.15 3.07 43.53 ± 0.90

The values presented are the mean value of three replicates ± standard deviation.

Table 2Seasonal change in biochemical properties of pulp of G. verrucosa .

Time of sampling Ash Total carbohydrates Reducing sugar Glucose

January 2009 16.17 ± 0.65 62.17 ± 1.26 34.83 ± 0.76 21.67 ± 0.58 May 2009 17.4 ± 0.85 63.50 ± 3.28 35.83 ± 0.76 24.00 ± 0.87 July 2009 17.4 ± 1.15 67.50 ± 1.50 37.67 ± 0.58 23.90 ± 1.68 January 2010 16.17 ± 0.65 63.50 ± 1.32 34.83 ± 0.76 21.67 ± 0.58 May 2010 17.4 ± 0.85 65.07 ± 1.50 35.93 ± 0.60 23.50 ± 0.87 July 2010 17.47 ± 1.12 66.67 ± 2.08 37.83 ± 0.29 24.80 ± 0.52

The values presented are the mean value of three replicates ± standard deviation.

Fig. 1. Enzymatic hydrolysis of algal pulp of G. verrucosa . (⁄The data points are the mean value of three replicates ± standard deviation.)

Fig. 2. Fermentation of enzymatic hydrolysate. (⁄The data points are the mean value of three replicates ± standard deviation.)

152 S. Kumar et al. / Bioresource Technology 135 (2013) 150–156

Table 3Comparison of saccharification and ethanol yield from various algal feedstocks using complete biomass.

Algae Parts used for saccharification and fermentation

Sugar released (g/gbiomass)

Ethanol (g/L)

Ethanol yield (g/gsugar)

Reference

Saccharina japonica Whole thallus 0.683 37.6 0.41 Adam et al. (2012)Sargassum

sagamianum Whole thallus NA 1–2 0.386 Hyeon et al. (2011)

Saccharina japonica Whole thallus 0.456 7.7 0.169 Jang et al. (2012)Kappaphycus alverzii Whole biomass plus carrageenan granule 0.306 20.6 0.39 Khambhaty et al.

(2012)Gelidium amansii Whole biomass 0.566 NA NA Kim et al. (2011)Laminaria japonica Whole biomass 0.376 23–29 0.41 Kim et al. (2011)Sargassum fulvellum Whole biomass 0.096 NA NA Kim et al. (2011)Ulva lactuca Whole biomass 0.194 NA NA Kim et al. (2011)Kappaphycus alverzii Whole biomass NA 6.8 0.369 Meinita et al. (2012)Gelidium amansii Whole biomass 0.422 27.6 0.38 Park et al. (2012)Gracilaria salicornia Two stage hydrolysis of fresh biomass 16.6 NA 0.079 Wang et al. (2011)Saccharina japonica Whole biomass 0.614 37.8 0.41 Wargacki et al. (2012)Sargassum

sagamianum Whole biomass NA 4–7 0.133–0.233 Yeon et al. (2010)

Laminaria digitata Whole biomass NA 5.61 NA Adams et al. (2011)

Algal Biomass (1000.0 kg)

Others(210 kg)

Moisture (45 kg)

Salt and Silt (215 kg)

Agar + Pulp (530 kg)

Agar (280 kg)

Pulp (250 kg)

Ash

(40 kg)

Lipid (20 kg)

Others (15 kg)

Protein (25 kg)

Holocellulose (150 kg)

Hemicellulose (50 kg)

Cellulose (100 kg)

Ethanol (38 kg)

Biomass (6 kg)

Agar extraction

Enzymatic hydrolysis

Fermentation

Cellulose (88 kg)

Unhydrolysed Cellulose (12 kg)

Others/Maintenance Energy (6 kg)

Fig. 3. Mass balancing of G. verrucosa during agar and ethanol production.

S. Kumar et al. / Bioresource Technology 135 (2013) 150–156 153

varied from 41.00 ± 1.0 to 43.53 ± 0.90, while the ash content of G.verrucosa ranged from 12.73 ± 0.21 to 13.97 ± 0.15% of dry weight from January 2009 to July 2010 (Table 1).

3.2. Change in biochemical properties in residual pulp of G. verrucosa

The residual algal pulp observed to have carbohydrat e content ranging from 63.50 ± 1.32 to 67.50 ± 1.50 (on percent dry weight

basis) from July 2009 to January 2010 (Table 2). While, the ash con- tent of pulp ranged from 16.17 ± 0.65 to 17.47 ± 0.85%. Maximum ash content of pulp was observed during January (17.4 ± 0.85%)and minimum during the month of July (16.17 ± 0.65%) (Table 2).There was a good variation in the ash content of the G. verrucosa .Reductio n in ash content in July may be due to a slight decrease in salinity because of heavier freshwater inflows during that peri- od. Prasad (1986) also observed reflect a decrease in ash content of

154 S. Kumar et al. / Bioresource Technology 135 (2013) 150–156

seaweeds due to reduction in salinity. Reducing sugar content of pulp varied from 34.83 ± 0.76 (January) to 37.83 ± 0.29% (July),whereas glucose content ranged from 21.67 ± 0.58 (January) to 24.80 ± 0.52% (July). This observation is also consisten t with the earlier report of Adams et al. (2011). Interestin gly, all the six samples exhibited a inverse correlation between carbohydrates and ash content and is in accordance with the earlier related work (Prasad, 1986 ).

Since, in all six pulp samples, the total carbohyd rate, reducing sugar and glucose concentration were recorded maximum, there- fore pulp obtained from algal mass collected in July was further used for enzymatic saccharification and fermentation.

3.3. Enzymatic hydrolysis of algal pulp of G. verrucosa

The time course of enzymatic saccharification of pulp of G.verrucosa exhibited a regular increase in sugars release (38.93 ± 0.76 g/L sugars) till 36 h, which remained almost constant thereafter (Fig. 1). The maximum rate of algal pulp saccharification (1.78 ± 0.07 g/L/h) was attained after 6 h of incubation and thereaf- ter it declined gradually showing the reciprocal relationship with saccharification efficiency similar to earlier reports (Gupta et al.,

Fig. 4. Schematic representatio

2009; Kuhad et al., 2010a ) (Fig. 1). The regular decrease in the rate of hydrolysis may be attributed to the end product inhibition of the enzymes by glucose and cellobiose (Kuhad et al., 1999 ). The opti- mal saccharification efficiency (87.58 ± 1.71%) was obtained after 36 h with a saccharification rate of 1.08 ± 0.02 g/L/h (Fig. 1). More- over the saccharification efficiency obtained in this study (�88%)was significantly higher than the saccharification efficiency of other seaweeds such as Ulva pertusa , Alaria crassifolia and Gelidiumelegans (Yanagisawa et al., 2011 ), and Kappaphyc us alverzii (Khambha ty et al., 2012 ), which may be because of more availabil- ity of fermentable carbohydrate in the Gracilaria pulp, optimum saccharification conditions and the enzyme efficiency. Compara -tively the enzymati c saccharification efficiency of G. verrucosa pulpis either more or comparable to saccharification efficiency of ligno- cellulosic s materials studied earlier (Adsul et al., 2005; Jeya et al., 2009; Gupta et al., 2011 ).

3.4. Fermentati on of enzymatic hydrolysate

Fermenta tion of enzymatic hydrolysates with S. cerevisiae produced maximum ethanol (14.89 ± 0.24 g/L) with yield (0.43 ± 0.01 g/g) and productivity (0.93 ± 0.02 g/L/h) after 16 h

n of Gracilaria biorefinery.

S. Kumar et al. / Bioresource Technology 135 (2013) 150–156 155

(Fig. 2). The ethanol yield from pulp of G. verrucosa (0.43 g/g sug- ars; 86% theoretical yield) was found comparatively better than earlier reports on using algal biomass for bioethanol production (Hyeon et al., 2011; Yanagisa wa et al., 2011 ). Hyeon and coworkers obtained an ethanol yield of 0.386 (g/g) using biomass of Sargas-sum sagamianum , while, Yanagisa wa et al. (2011) when used bio- mass of U. pertusa , A. crassifolia and G. elegans for ethanol production reported an ethanol yield of 0.381, 0.281 and 0.376 g/ g, respectively . Table 3 depicts a comparison of bioethanol produc- tion with other algal. Interestingl y, the ethanol yield from G. verru- cosa was also found comparable with the previousl y reported ethanol yields from various lignocellulos ics materials such as corn- cob (0.48 g/g sugars; 96% theoretical yield) (Chen et al., 2007 ) Pros-opis juliflora (0.49 g/g sugars; 98% theoretical yield) (Gupta et al., 2009), Lantana camara (0.48 g/g sugars; 96% theoretical yield) (Ku-had et al., 2010a ) and newspaper waste (0.39 g/g sugars; 78% the- oretical yield) (Kuhad et al., 2010b ). The yeast used here produced maximum ethanol from enzymatic hydrolysate of G. verrucosa after16 h of fermentati on and it declined thereafter (Fig. 2). The decline in ethanol production after 16 h fermentation could be attributed to consumptio n of accumulate d ethanol by the organism as has been observed during our earlier studies (Gupta et al., 2009; Kuhad et al., 2010a ). According to Ramon-P ortugal et al. (2004), when the ethanol accumulated in the medium, the microbial population was adapted to consume simultaneou sly sugar and ethanol.

3.5. Mass balance

Mass-energ y balancing was used advantageousl y in analyzing experimental results and in the operation of fermentation pro- cesses. Here, we have extrapolated our results to 1000.0 kg level to make the study more comprehensive . The results revealed that out of 1000 kg algal biomass �280 kg agar (�53% of total carbohy- drate) could be produced and the leftover material as pulp ob- tained was �250 kg (47% of total carbohydrat e). The pulp contained �60% holocellu lose (comprising 40% cellulose and 20% hemicellulos e) (Fig. 3). On enzymatic hydrolysis approximat ely 88% of cellulose fraction was converte d into sugars (mostly glu- cose), which was subsequent ly fermented to ethanol with an eth- anol fermentation efficiency of 86% (Fig. 3). This showed that the ethanol production process has good efficiency and the process seems to be commercially attractive.

3.6. Integrated seaweed biorefinery: future prospects

Bioethanol production from seaweeds will reduce the usage of fresh water, fertilizer s and agricultural land. A schemati c diagram of Gracilaria biorefinery is shown in Fig. 4. Average worldwide Gracilaria production including wild and cultivated is 1.4 million tons of fresh weight (FAO, 2010 ) and the present agar production is 9600 tons, which costs �US$ 115,200 (Bixler and Porse, 2010 ).The algal pulp is usually disposed off as a waste and eventual ly produces fouling smell in the surroundings. The pulp being rich in cellulose can be utilized for as raw material for bioethanol pro- duction. Even after the ethanol production the leftover residue still contains good amount of organic matter and useful minerals, and eventually could be used as biofertilizer. In addition, it has been observed that seaweeds can efficiently absorb CO 2 from the seawa- ter. Thus, carbon sequestra tion from the atmosph ere will lower down the acidification of the ocean as well (Chung et al., 2011 ).

4. Conclusion

The present study demonstrates that Gracilaria production if managed properly, a biorefinery could be developed.

Acknowled gements

The authors gratefully acknowledge Universit y Grant Commis- sion (JRF/RGNF/SC/F3/2007-08 ) and Council of Scientific and Indus- trial Research, New Delhi (CSIR-SRF 9/45(1078)/2011-EMR-I) for the financial support given. RCK and DBS are specially grateful to Universit y of Delhi for financial support.

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