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Accepted Manuscript

Production of biodiesel and bioethanolusingalgal biomass harvested from fresh


water river

Vinod Kumar, Manisha Nanda, H.C. Joshi, Ajay Singh, Sonal Sharma, MonuVerma

PII: S0960-1481(17)30977-1

DOI: 10.1016/j.renene.2017.10.016

Reference: RENE 9304

To appear in: Renewable Energy

Received Date: 18 March 2017

Revised Date: 24 August 2017

Accepted Date: 05 October 2017

Please cite this article as: Vinod Kumar, Manisha Nanda, H.C. Joshi, Ajay Singh, Sonal Sharma,
MonuVerma, Production of biodiesel and bioethanolusingalgal biomass harvested from fresh water
river, Renewable Energy (2017), doi: 10.1016/j.renene.2017.10.016

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Highlights

 Fresh water macroalgal biomass can be utilized for production of liquid biofuels.
 18.6% of lipid was obtained from macroalgal biomass.
 Addition of diesel and butanol to algal biodiesel can improve engine performance
 Emission characteristics of CO and NOx can be improved by adding butanol.
 Theoretical yield of ethanol was estimated 61.0% from lipid extracted algal biomass
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1 Production of biodiesel and bioethanolusingalgal biomass harvested from fresh water river

2 Vinod Kumara*, Manisha Nandab, H.C. Joshia, Ajay Singha, Sonal Sharmaa and MonuVermaa

4 aDept. of Chemistry, Uttaranchal University, Dehradun, India

5 bDept. of Biotechnology, Dolphin (PG) Institute of Biomedical and Natural Sciences, Dehradun,

6 India

8 Corresponding Author:-

9 Dr Vinod Kumar ,

10 Dept. of Chemistry,

11 Uttaranchal University,

12 Dehradun, India

13 Email: vinodkdhatwalia@gmail.com

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22 Abstract

23 In this study, an integrated biomass conversion concept of producing liquid biofuels from fresh

24 water macroalgal biomass was investigated. The algal biomass was collected from the Song

25 river, Dehradun, Uttarakhand, India and processed under laboratory. 0.650 g dry wt m−2of algal

26 biomass was harvested from the freshwater river. The collected algal biomass contained mainly

27 2 macoalgae species. Lipid extraction was done by soxhlet extraction method using chloroform:

28 methanol (2:1) as solvent. 18.6% of lipid was obtained from macroalgae biomass. Blends of

29 microalgae biodiesel with, butanol and diesel fuel (A5B25D70 and A10B30D60) were prepared

30 by Inline blending method on a volume basis. Oil extracted algal biomass was further hydrolyzed

31 for release of fermentable sugar. The theoretical yield of conversion of fermentable sugars to

32 bioethanol was estimated and found to be 61.0%.

33 Keywords: Algae; biomass; biodiesel; bioethanol

34 1. Introduction

35 With a large area, abundant freshwater resources, complex topography and diverse climates,

36 India is one of the countries with the greatest diversity of algal resources. There are diverse and

37 abundant freshwater algae in India. Macroalgae have diverse biomass (oxygen-containing

38 organic components and inorganic minerals) applications as a source of food and biofuels [1].

39 The majority of research focuses on marine macroalgae (seaweed) and no significant production

40 of freshwater macroalgae exists [2]. Freshwater macroalgae viz., Cladophora, Enteromorpha,

41 Hydrodictyon, Microspora, Mougeotia, Oedogonium, Rhizoclonium, Spirogyra, Tribonema,

42 Ulothrix,Vaucheria and Zygnema are biological resource for various valuable compounds such

43 as protein, carbohydrates and lipids [3]. Macroalgae can form dense floating mats on water

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44 surfaces. This allows for efficient and cost-efficient biomass harvesting as compared to

45 dewatering an equivalent biomass of suspended microalgae. Limited research has been done on

46 freshwater macroalgae as a biofuels feedstock [4]. So for the in this study we have used this

47 mixed macroalgal biomass for the production of liquid biofuels (biodiesel and bioethanol).

48 Cellulose and hemicellulose can be hydrolysed into simple sugars either enzymatically or by acid

49 hydrolysis for producing bioethanol.The success of the bioethanol production from algal biomass

50 mainly depends on the amount of its carbohydrate contents [5]. R. Trivedi et al., [6] reported

51 about 11% of cellulose in macroalgal biomass which can be further hydrolyzed to simple sugar. .

52 The hydrolysis product being a six carbon sugar can be easily fermented to ethanol [7].

53 This study has two specific objectives, which are as listed as follows: The first is extraction of

54 lipids from mixed algal biomass. Second is to convert the lipid extracted algal biomass into

55 fermentable sugars for the production of bioethanol.

56 2. Materials and Methods

57 2.1 Materials

58 All solvents and reagents used in this study were HPLC grade. Standard for TLC (Triolein) was

59 acquired from Sigma Aldrich (St.99 Louis, MO, USA). All solvents and reagents used in this

60 study were HPLC grade. Cellulase enzyme was purchased from Sigma Aldrich (St. Louis, MO,

61 USA).

62 2.2 Water measurements and harvesting of wild algal biomass

63 Microalgal biomass was collected from freshwater Song river, Dehradun, India in the month of

64 November-December 2016, when water level was 30-35 cm and flow rate was slow. During the

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65 harvesting of macro algal biomass water samples were also collected in clean glass bottles for

66 the measurements of different water characteristics.

67 2.3 Morphological characteristics of the collected Algal biomass

68 Macroalgal biomass was identified based on the morphological characteristics. Species were

69 examined under dissecting and compound light microscopes and their morphological

70 characteristics were recorded.

71 2.4 Determination of lipid content and lipid productivity

72 For lipid extraction soxhlet was used. In this extraction method 25g of algae biomass was

73 processed with 250 ml of chloroform: methanol (2:1) for 6 h in soxhlet system. The weight of

74 oily extract was weighed and counted as oil content (% DW).

75 Lipid content= (Final lipid extracted- Initial lipid)/DCW.

76 Lipid productivity= Lipid content X Biomass productivity/100.

77 For triacylglycerols detection lipid sample (5 μl) was spotted on 0.25-mm-thick silica gel for

78 TLC plate and TAGs were visualized according to N. Arora et al., [8].

79 2.6 Transesterification, fatty acid profile and biodiesel properties

80 The total extracted lipids were transesterified into fatty acid methyl esters (FAMEs) by

81 methanolic sulphuric acid (6%) [9]. The FAMEs were analyzed using gas chromatography- mass

82 spectroscopy (GC-MS; Agilent technologies, USA), with electron ionization (70 eV), DB-5

83 capillary column (30 mm 0.25 mm 1μm) and helium (1ml/min) as carrier gas. 1 μl of of sample

84 was injected and completed according to N. Arora et al. [8].

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85 The physicochemical properties (iodine value, saponification value, specific gravity, acid value,

86 cetane number, high heating value, long chain saturation factor, specific gravity ) of the biodiesel

87 obtained from macro algal biomass were determined according to ASTM D-6571 specifications.

88 Fire and flash point were determined by Pensky-Martens closed cup tester.

89 2.9. Four stroke engine test of biodiesel

90 A four stroke engine, cylinder was used in this study. The Specification of engine is given in

91 table 1. Before starting the experiments the test engine was cleaned by fresh diesel fuel. Pure

92 algal biodiesel cannot be used directly to the engine due to lower oxidation stability, low cetane

93 index and low calorific value. So before conducting the engine test, blending of algal biodiesel

94 was done. Two blends of algae biodiesel with diesel and n- butanol were prepared, namely

95 A5B25D70 (5% algae, 25% Butanol and 70% diesel) and A10B30D60 (10% algae, 30% Butanol

96 and 60% diesel). Engine performances were read with the help of dynamometer with control unit

97 having a torque range of 0–1700 Nm and speed range of 0–7500 rpm to measure engine torque.

98 Table 1: Specifications of the test engine.

Type Specification

Make Mitsubishi Canter, 4D34-2A

Cycle four stroke

Cylinder Four

Type Direct injection diesel with glow plug

Displacement 3907 cc

Bore 104 mm

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Stroke 115 mm

Power 89 kW @ 3200 rpm

Torque 295 Nm @ 1800 rpm

Oil Cooler Water cooled

Weight 325 kg

99

100 2.8 Release of fermentable sugars

101 The lipid extracted macroalgal biomass was dried in open field. When there was no solvent odor

102 from algae biomass, indicating acceptable evaporation of solvent, this dried lipid-extracted algal

103 biomass was used for release of fermentable sugars.

104 2.9 Acid hydrolysis

105 Acid hydrolysis was done by single step hydrolysis method. 25g lipid-extracted algal biomass

106 was mixed with 250 ml of 1, 5 and 10 % of two acids HCl and H2SO4 at 3 different

107 concentration acids. Conical flasks were placed into autoclave for 2 h at 121 °C and 15 psi.

108 Hydrolysates were cooled at room temperature and then neutralized with 10M NaOH to pH 7.

109 The residual biomass was separated from the hydrolysates by centrifugation at 8000 rpm for 15

110 min. The supernatant was used for sugar and other analyses.

111 2.11 2 Enzymatic hydrolysis

112 Enzymatic hydrolysis was performed by according K. Karthika et al., [10]. Fine slurry was

113 prepared by adding distilled water to 10g lipid-extracted algal biomass powder, then

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114 supplemented with cellulase enzyme (Trichoderma reesei ATCC 26921) of 66 FPU g−1 dry

115 matter. The residual biomass was separated from the hydrolysates by centrifugation at 8000 rpm

116 for 15 min. The supernatant was used for sugar and other analyses.

117 2.12 Sugar concentration analysis

118 The total reducing sugars were determined by colorimetric and chromatographic method.

119 Colorimetric method was performed according to G. L. Miller [11]. For the optimization

120 experiments, sugar concentrations in the samples were quantified by high pressure liquid

121 chromatography (HPLC) [12].

122 2.12 Separate hydrolysis and fermentation

123 For the fermentation with S. cerevisiae, 0.5 g of dry yeast was added as inoculum to 100 mL of

124 hydrolyzates. Fermentation was carried out at 32°C and pH 5 for 7 days with agitation at 150

125 rpm. Distillate obtained from rotary evaporator was used to determine bioethanol concentration

126 colorimetrically using potassium dichromate method [13].

127 The maximum theoretical ethanol yield was calculated as follows [14]:

128 Ymax (%) = Ethanol produced in reactor (g)


129 ___________________________ ×100
130 Initial sugar in the reactor (g) ×0.511

131 2.13 Fermentation inhibitors

132 During the acid hydrolysis of algae biomass, various types of chemicals generated that act as

133 fermentation inhibitor. Fermentation inhibitory compounds were evaluated using HPLC

134 technique [12].

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135 2.14 Statistics

136 The statistical analysis was carried out by analyzing the triplicate (n=3) results. The variation of

137 lipid and sugar contents was investigated by One-way ANOVA using Graph Pad Prism software

138 (version 6.0f) with p < 0.05 was carried out for data validation.

139 1. Results and Discussion

140 3.1 Water characteristics, biomass Production & Identification

141 The results of river water parameters, such as pH, salinity, temperature, BOD etc, are given in

142 table 2. We have harvested 0.650 g dry wt m−2of algal biomass from Song river, Dehradun,

143 India, dry mass factor was 5%. We have indentified 2 macro algae Spirogyra and Oedogonium

144 from collected algal biomass.

145 Table 2: Characteristics of the Tons river water used in this study.

Parameters Value

Temperature 17.4±0.1

PH 7.7±0.3

TDS (mg L_1) 420±0.3

BOD(mg/l) 4.9±0.2

DO 10±1.2

Alkalinity (mg/l) 189±0.2

Hardness (mg/l) 580±0.2

146

147 TDS= Total dissolved solid, DO= Dissolved oxygen, BOD= Biochemical oxygen demand. The data are mean ± S.D. for

148 triplicate (n=3) results (p < 0.05).

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149 3.4 Physicochemical property analysis of fatty acid and Biodiesels

150 The presence of TAGs in the total extracted lipids was confirmed using TLC. 18.6 % of lipid

151 was obtained from the collected algal biomass. GC-MS analysis of FAMEs revealed that

152 saturated fatty acid were the major fatty acids obtained (C16:0). Mono saturated fatty acids

153 C16:1, C18:1 and C20:1 were also present (Table 3, fig. 1). This may be due to thermo-

154 degradation of long chain polyunsaturated fatty acids during soxhlet extraction [15]. R. Halim et

155 al., [16] reported that fatty acids mainly C16 hexadecanoic (or palmitic) acid, C18:1 (n-9) oleic

156 acid and C18:2 (n-6) octadecadienoic (or linoleic) acid are normally treated as the major

157 components for microalgal biodiesel production.

158

159

160 Fig:1 GC-MS analysis of FAMEs.

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161 Table 3: Fatty acid composition of wild algal biomass by using different solvents

162

163
Solvent Fatty acid composition (wt%) SFA MUFA

164 C12: C1 C1 C16: C18: C20:

1 4 6 1 1 1
165 3.5 Prediction of
chloroform: 4 5 48 10 19 2 60 20
166 Biodiesel
methanol (2:1)
167 Properties

168 FAMEs obtained from macroalgal biomass has combustion properties similar to conventional

169 diesel fuel. Important parameters of biodiesel are cetane number, and Iodine value which

170 determine the combustion behaviour, quality of biodiesel, stability and performance are

171 summarized in table 4.

172 Table 4: Comparison of physical properties of different FAMEs obtained from algal biomass

173 with plant oil methyl esters (JPE, PME ) and commercial biodiesel

Physical Plant oil Algal ASTM EN 14214 Commercial

properties methyl FAME D6751-02 biodiesel

esters

JME PME

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174 Saponification 187 49.56 130.10 - -

175 Value(mg

176 KOH)

Iodine value 54 61 38.02 - 120 (maxi) 130


177

(g I2/100g)
178

Specific - - 0.756 - - -
179
gravity (kg−1)
180 Acid Value - - 2.5 0.8 - 0.50

181 mg KOH g-1

Flash point 180 - 45 93.0 Min - 35


182
Fire point 256 - 49 - - -
183 3.6
Cetane Value 40 30 47 (min) 51(min) 47
184 Engin
High Heating - 41.00 -
185 e
Value
186 perfor
Long chain - - 57 - - -
187 mance
saturation
188 and
factor
189 emissi
(% wt)
190 on
Cold flow -2 13 5 - - -5
191 charac
plugging
192 teristi
property
193 cs
(0C)

194 3.6.1 Brake Power and Torque Output:


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195 The maximum torque was obtained at about 1900 rpm for all blended test fuel. The average

196 torque values of blended biodiesel of A10B30D60 and A5B25D70 decreased approximately by

197 6% and 5% respectively. At 2200 rpm of maximum brake power was obtained for all tested

198 blended fuel. Generally brake power output values of both the algal based biodiesel with blends

199 of diesel were reduced in the addition of butanol as compared with diesel fuel. Fig 2 and 3 shows

200 the variations torque and brake power with respect to test fuels. Rahim et al., [17] reported that

201 brake torque for diesel fuel is higher than biodiesel fuel. At low speed, the brake torque is higher,

202 as engine speed increase further, torque decreases. The torque decreases because the engine is

203 unable to ingest a full charge of air at the higher speed [18]. Heat content of the fuel blend

204 decreases with the increasing amount of biodiesel compared to that of diesel fuel [19, 20]. The

205 brake torque level increased with increased the proportion of biodiesel due to high lubricity and

206 the higher oxygen content of biodiesel [21, 22] Brake torque and brake power is also decreases

207 when compared to the diesel fuel due to presence of oxygencontents of algal biodiesel and

208 butanol [23].

209

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300

250

200
Torque (Nm)

150

100

Diesel
50 A5B25D70
A10B30D60

0
500 1000 1500 2000 2500 3000
Engine Spped (rpm)

210

211 Fig. 2. Torque output versus engine speed for the test blended fuel

60

50
Brake Power (KW)

40

30

20 Diesel
A5B25D70
A10B30D60
10
800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
Engine Speed (rpm)

212

213 Fig.3. Brake power output versus engine speed for the test blended fuels.

214 3.6.2 Brake specific fuel consumption (BSFC)

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215 Figure 4 shows the variation of brake specific fuel consumption (BSFC) of diesel and blended

216 biodiesel. When two different fuels are blend, the fuel consumption may not be reliable due to

217 heating and density differences of the two fuels [23]. For different blended fuels followed by the

218 decreases in BSFC with the increases in an engine speed and brake power which indicates the

219 minimum role of oxygen at maximum engine speed [24]. The highest BSFC was for

220 A10B30D60 while the lowest BSFC was obtained from diesel for all speeds of the engine. The

221 high BSFC is due to low heating values of the algal biodiesels than diesel fuel [20]. The BSFC

222 value increases about 5.3% and 8.7% for A5B25D70 and A10B30D60 respectively. The values

223 for the BSFC increase with the increasing amount of biodiesel in blended fuels [22]. This is due

224 to the produced lower brake power caused by the lower energy content of the biodiesel.

225 [20,27,28]. The calorific value of n-butanol is quite low and expected that the higher value of BSFC

226 (A10B30D60 and A5B25D70). Therefore, on the addition of butanol increases the fuel consumption

227 for biodiesel diesel blends (A10B30D60 and A5B25D70) Compared to diesel fuel. [28]. B.D.

228 Wahlen et al., [29] reported that biofuels showed higher BSFC across the rpm range of the

229 test compared to diesel which is consistent with their lower energy content.

230

231

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600

500
BSFC (g/KWH)

400

300

200
Diesel
100 A5B25D70
A10B30D60

1000 1200 1400 1600 1800 2000 2200 2400


Engine Speed (rpm)
232

233 Fig. 4. Comparison of BSFC values for the test fuels.

234 3.6.3 NOx emission

235 It has been generally observed that biodiesel increases emissions for NOx relative to diesel [30].

236 Similar results were reported by other authors in the literature [31,32]. The variations of NOx

237 emission of different blends fuel are presented in fig 5. NOx values were decreased with the

238 addition of butanol to the blends as compared to diesel. Formation of NOx depends on the

239 availability of excess oxygen. Because of the emission of NOx there was increase in biodiesel

240 blends as compared to diesel (Fig.5). With addition of butanol to biodiesel NOx emissions

241 decrease [32,33]. Formation of NOx is affected by the higher combustions [34]. Biodiesel

242 contain higher oxygen content which react with the nitrogen component in the surrounding air,

243 resulting in a higher emission of NOx [33]. Wahlen et al., [29] reported that NOx emissions for

244 algae biodiesel were lower than for diesel. McCormick et al. found that NOx emissions depends

245 upon the types of fatty acid, it increased with increasing amount of unsaturated fatty acids.

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246 Additional studies have found that low polyunsaturated fatty acids and shorter chain length fatty

247 acids containing oils contribute to lower NOx emissions than soybean biodiesel [35].

248

249

1000

800

600
Nox (PPM)

400

Diesel
A5B25D70
200 A10B30D60

0
800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
Engine Speed (rpm)
250

251 Fig. 5: Comparison of NOx emissions for the blended test fuels.

252 3.6.4 CO emissions

253 CO emission is generally lower in biodiesel than the diesel fuel, because biodiesel contains more

254 oxygen and this oxygen enhances the complete combustion of fuel. Butanol blends to biodiesel

255 also decrease the CO emission. As can be seen in Figure 6, that on with increase the

256 concentration of biodiesel and n-butanol the CO emission decrease. A10B30D60 showed less

257 emission as compared to A5B25D70 and diesel. Other authors, also reported that decrease in

258 CO emissions occurs when substituting diesel fuel with biodiesel and alcohol [36-37]. One of

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259 the reasons for the less emission of CO with the blends of butanol may be due to lower C/H ratio.

260 [37]. Altun et al., [38] reported that fuel-borne oxygen is more effective for reducing CO

261 emissions than the external oxygen supplied with the air. In contrast to our results, some authors

262 reported that CO emission decreased with the increase in engine speed [39,40]. This is because

263 the air-fuel mixing process may become more intensive and increase the conversion of CO to

264 CO2 [41].

265

400

350
CO (ppm)

300

250

Diesel
A5B25D70
200
A10B30D60

1000 1500 2000 2500 3000


Engine Speed (rpm)
266

267 Fig. 6: Comparison of CO emissions for the blendedtest fuels.

268 3.7 Release of fermentable sugars

269 From lipid-extracted algal biomass single-step acid hydrolysis should be capable of releasing

270 high yield of fermentable sugars (Table 5). In this sulfuric acid and hydrochloric acid at 03

271 different concentrations were used. H2SO4 and HCl are widely used for treating lignocellulosic

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272 materials, because they are powerful agents for cellulose hydrolysis [42]. Acids like hydrochloric

273 acid and nitric acid have also been examined for pretreatment of various lignocellulosic materials

274 [43]. By using single-step method (1210C and 15 psi for 2 h) maximum yield of sugar 230.02±03

275 mg/g of lipid extracted biomass was reported by 10 % H2SO4and minimum was 10% HCl

276 212.24±03 (Table 5, Fig. 7). M. Idrees et al. [44] has reported high reducing sugar yield when

277 plant material was pretreated at 121 ºC. G. L. Miller et al., [11] reported one-step acid hydrolysis

278 method gave best results as compared to conventional two-step approach for different biomass.

279 Table: 5 Release of fermentable carbohydrate (mg/g algae) by single-step hydrolysis.

% of HCL H2SO4

acid

used

1 10.02±02 11.15±02

5 70.03±02 89.01±01

10 212.24±03 230.02±03

280 The data are mean ± S.D. for triplicate (n=3) results (p < 0.05).

281

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300

250
mg/g algae

200

150
HCL
100 H2SO4

50

0
0 30 60 90 120 150
Time (minutes)
282

283 Fig.7: Release of sugars concentration during single-step hydrolysis.

284

285 3.8 Fermentation and Inhibitor

286 In this study S. cerevisiae was added to all the hydrolysates produced by different methods [45].

287 Only sulfuric acid hydrolysate showed the growth of S. cerevisiae cells. Hydrochloric acid

288 does not showed the growth of S. cerevisiae due to the presence offermentation inhibitors.

289 Fermentation with Saccharomyces cerevisiae on the concentrated hydrolysates produced 31.6%

290 of bioethanol yield by single- step acid hydrolysis. The fermentation inhibitors were generated

291 during different carbohydrates hydrolysis processes. These inhibitory compounds are generally

292 furan derivatives, weak acids, and phenolic compounds [46]. In this study, the presence of

293 common inhibitors such as acetic acid, furans, levulinic acid, and formic acid were examined in

294 the hydrolysates obtained by acids (Table 6). Our study supported the finding of M. Mirsiaghi

295 [12], who reported similar inhibitors.

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296 Table:6 Detected inhibitors and their corresponding yields in terms of mg inhibitor/g algae

297 biomass. NR-Not reported.

Treatment Levulinic acid Acetic acid Hydroxymethylfurfura

1% HCl NR 2.1±02 NR

5% HCl 1.43±01 3.9±01 NR

10% HCl 3.9±01 7.9±01 0.15±03

1% H2SO4 NR 1.10±03 NR

5% H2SO4 0.10±02 2.7±01 NR

10% H2SO4 0.72±01 4.2±02 NR

298 The data are mean ± S.D. for triplicate (n=3) results (p < 0.05).

299 3.9 Total renewability of algal biomass

300 The total yield of lipid was estimated 18.6 % of dry biomass. Conversion of biomass into

301 fermentable sugars was estimated to 23%. Total renewability of algal biomass in this study was

302 41.6% of dry algal biomass.

303 4. Conclusion

304 In this study, an integrated biomass conversion concept of producing liquid biofuels from fresh

305 water macroalgal biomass was investigated. The algal biomass was collected from the Song river

306 Dehradun, India and processed for liquid biofuels production in laboratory. 18.6% of lipid was

307 obtained from macroalgae biomass. FAMEs profile was analyzed according to ASTM D6751

308 specification and comparable to plant oil methyl esters. Characterization of performance and

309 emission characteristics of biodiesel was conducted on a four stroke and four-cylinder diesel

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310 engine by blending it with butanol and diesel fuel. The test results indicated that A10B30D60

311 (10% algae, 30% Butanol and 60% diesel) showed good performance efficiency and

312 environmentally friendly emissions.The emission characteristics of CO and NOx were improved

313 with the addition of butanol. On the addition of butanol to the algal biodiesel brake power and

314 torque output of engine was slightly reduced. From the above investigation we have concluded

315 that the diesel and butanol can be used as an auspicious accompaniment to algal based biodiesel

316 in conventional diesel engine. Oil extracted algal biomass was further hydrolyzed for release of

317 fermentable sugar. The theoretical yield of conversion of fermentable sugars to bioethanol was

318 estimated and found to be 61.0 %.

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