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R. Sen, RSC Adv., 2016, DOI: 10.1039/C6RA01477A.
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DOI: 10.1039/C6RA01477A
5 India
7 Abstract:
10 necessary to make a good value proposition. Present study thus focuses on the development of an
11 integrated downstream processing strategy for the concomitant extraction of lipid and
13 90.62.8% and 98.72.1% was accomplished for Chlorococcum cells by employing minimal
14 FeCl3 in first 30 min and 150 min, respectively, as opposed to 55.522.2% in 30 min by own
15 self-flocculation ability. Various physical and chemical pretreatment methods were attempted to
16 maximize the recovery of sugars and lipids, separately and simultaneously. Microalgal lipid was
17 efficiently recovered by cell disruption with bead-beating and using chloroform-methanol (2:1,
18 v/v) as extraction solvent. The microalgal carbohydrate recovery and conversion into free
19 fermentable sugars was found to be greater in case of acid hydrolysis as compared to alkaline
20 hydrolysis. Microalgal biomass, when pretreated separately, resulted in total sugar yield of
21 89.63.1% and total lipid yield of 96.22.9%. Therefore, to improve the process performance,
22 simultaneous extraction of carbohydrate and lipid was carried out by bead-beating followed by
1
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23 acid treatment. The recovery of fermentable sugars from supernatant and that of lipid from pellet
24 were most efficient with the respective yields of nearly 86.52.6% and 74.11.8%, without any
25 downtime. The extracted lipid was then converted into fatty acid methyl esters (FAME) as
29 obtained. Thus, the study holistically addresses some technological challenges in the downstream
30 processing of microalgal biomass for efficient recovery of lipid and carbohydrate for production
34
35 1. Introduction:
36
37 The continued use of fossil-derived liquid fuels is regarded as unsustainable due to their
38 depleting reserves and associated environmental concerns.1 Almost 85% share of all petroleum-
39 derived fuels is burned-out in the transportation sector.2 Global energy utilization is expected to
40 increase manifold due to extensive anthropogenic activities, coupled with the rising population.
41 Therefore, there is a need for sustainable initiatives that not only lessen the dependence on fossil
42 fuels but also solve the climate crisis. Microalgal feedstock, a credible biofuels source, has
43 drawn much attention as a renewable and sustainable alternative to rapidly depleting fossil fuels,
44 This is because many microalgal species have superior photosynthetic efficiencies and biomass
45 production potentials than terrestrial crops. Microalgae also have environmental advantage as
46 they can grow in a wide range of pH, nutrients availability, temperatures, wastewater, etc., and
2
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47 most importantly microalgal has short generation cycle due to which it can be harvested year-
48 round.3,4 Microalgal derived lipids and carbohydrates could be efficiently used for biofuels
49 production such as biodiesel and bioethanol, and are considered potential liquid biofuels because
50 of their compatibilities with the existing transportation systems and fuel markets.57 Besides
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53 for bioethanol production. Bioethanol from microalgae displays greater sustainability and
55 microalgae have been explored for biofuels production, such as Chlorella sp., Botryococcus sp.,
56 Scenedesmus sp., Nannochloropsis sp., Chlorococcum sp., etc.1,7,10,11 Chlorococcum sp. is one of
57 the economically important microalgal species that is rich in bioactive substances including
58 carotenoids, eicosapentaenoic acid, fucoxanthin, etc. and has been widely used in
59 aquaculture.10,12,13 Chlorococcum sp. has also shown potential to be useful biofuel feedstock, as
60 lipid and carbohydrate source, for biodiesel and bioethanol production.9,14 Microalgae derived
61 biofuels may prove to be an alternative fuel source that can be developed for the long-term
62 replacement of rapidly depleting fossil fuel. In addition to their potential biofuels applications,
63 microalgae have been applied in CO2 sequestration, wastewater bioremediation and production
65 Despite various benefits and significant progress made in the development of the algal biofuels
66 generation, some technological drawbacks remain while using algal feedstock in the downstream
67 processing for conversion of algal biomass into biofuels, which subsequently diminishes the
68 economics of fuel production.16 Major practical limitation for the algal biofuel industry has
69 remained in the extraction of biofuels from wet algal biomass. Moreover, most of the reported
3
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70 literature have focused on upstream processes, i.e., algal cultivation technologies.4 Typically, the
71 biodiesel production from microalgae involves four steps such as cultivation, harvesting, lipid
72 extraction and transesterification. With the exception of cultivation, the downstream processes
73 constituting the last three steps contribute to approximately 60% of the total biodiesel production
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76 could vary based on the type of harvesting technology used, nature or types of microalgae and
77 the density of the microalgal culture.18 Concentrating methods play an important role to increase
78 the solid concentration of microalgae in suspension and volume reduction, which could
80 processes involve the use of flocculants (chemical and biological based), centrifugation, gravity
82 microbial flocculation.19,20 Flocculation has lower energy requirements than centrifugation, and
83 increases the settling rate by congregating suspended particles to increase the biomass
85 for the effective extraction of lipids to augment biofuel yields.15 This pretreatment, besides being
86 quite energy-intensive step, involves large amount of chemicals, such as an acid, base, organic
87 solvents and/or physical treatment including cell disruption, autoclaving, sonication, etc., either
88 incorporated in stepwise or simultaneous mode.15 Lipid recovery is often limited by the low
89 extraction yields from the algal cells.21 Even bioethanol production from algal biomass requires
90 pretreatment step to make the sugars accessible for fermentation.5 Finally, in the product
91 conversion step, the extracted microalgal lipid is converted into the final product as fatty acid
93 biodiesel is conventionally catalyzed by catalysts, such as acid, alkali, solid catalyst or enzymes.
94 This final stage also accounts the major cost to the overall biodiesel production process.22
96 technologies that have a potential to be integrated into the existing upstream and downstream
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98 processing steps (such as, harvesting, lipid extraction, and transesterification) when performed
99 independently demands different chemicals and/or equipments, which account for the high
100 processing cost.15 Additionally, most of the current algal biofuels technologies, adopt one strain
101 and one product specific strategy, where mainly lipid fraction serves as a feedstock for biodiesel
102 production. Nevertheless, the key practical barrier for the algal biofuel industry is achieving a net
103 positive cost and energy balance for the extraction of biofuels from wet algal biomass.4 Hence, it
104 is necessary to improve the economics and sustainability of the biofuel production process,
105 through various strategies like, improving the efficiency of downstream processing by
106 integration of various unit operations, combining additional value added co-products to biofuel
107 products, etc. Integration of various biomass components as well as involving technologies in the
108 form of biorefinery model presents an immense opportunity to advance in the field of microalgal
109 based biofuels production. Moreover, it is reported that the production of more than one product
110 improves the economic efficiency by 33%, compared to one strain and one product specific
111 process.23
112 Currently, there are large number of reports on independent production of bioethanol and
113 biodiesel from microalgae, however not many studies that reports an integrated process of
114 bioethanol and biodiesel production, except few studies carried out by Wang et al.,8 and Laurens
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115 et al.23 There are barely any reports that have demonstrated a systematic downstream processing
116 of microalgal culture, starting from harvesting to pretreatment of algal biomass for effective
117 extraction of lipid and carbohydrate in an integrated approach, and finally their conversion into
118 biofuels, biodiesel and bioethanol. The objective of this work was to present a holistic approach
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119 to downstream processing for the conversion of algal biomass into biofuel products such as
123 The microalgal species used in this study was a Chlorococcum infusionum.14 The microalga was
124 cultivated in airlift photobioreactor (APBR) with working volume of 1.5 L (length: 1180 mm,
125 diameter: 75 mm) and supplied with air at a flow rate of 1500 mL min-1 at 302 C under
126 illumination of 100 mol photons m-2s-1 (14:10 h light:dark cycle) to produce microalgal
127 biomass. The microalgal slurry was collected from the early stationary phase grown culture for
128 the usage in downstream processing for harvesting studies. The composition of algae biomass
131 Harvesting studies using FeCl3 and Al2SO4 as flocculants on the microalgae culture was carried
132 out in cylindrical glass tubes with 25 mL of culture volume. After addition of flocculants, the
133 algal suspension was rapidly mixed and left for settling. The doses of flocculants were varied
134 from 10 to 100 mg L-1. In the subsequent experiments, effect of change in microalgal culture
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135 densities (0.251.5 gL-1) was also determined. The harvesting efficiency was calculated
137 (1)
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138 where, ODi and ODf correspond to optical density of sample taken at initial time zero and at final
143 Cell disruption of the algal biomass by various physical and chemical means such as bead-
144 beating, homogenization, sonication, lyophilization, acid treatment and autoclaving for
145 extraction of lipid was conducted. The lipid was then directly extracted using chloroform and
148 Various single and binary solvent systems were selected for the extraction of lipid for this study.
149 Lipids were extracted in four various single solvent systems, viz. chloroform, hexane, methanol
150 and ethanol (Merck, India), at room temperature. Binary solvents system such as chloroform-
151 methanol (in 1:2 and 2:1 ratio), hexane-methanol (1:1), and hexane-ethanol (1:1) were also used
152 for lipid extraction. The extracted lipid was filtered and lipid content was quantified
153 gravimetrically following evaporation of the solvents, and was expressed as % dry cell weight
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156 Studies on chemical pretreatment of microalgae for the extraction of sugars was carried out by
157 investigating different physicochemical parameters such as different chemicals (acid/base), their
158 concentration, temperature and pre-treatment time. The lyophilized and grounded microalgae
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were pretreated with various acid (H2SO4 and H3PO4) and alkali (NaOH and ammonium)
160 chemicals. Further, the effects of different concentration of chemicals (NaOH and H2SO4)
161 ranging from 0.1 to 0.3M were evaluated. Next, the effect of temperature varying from 70 to 120
162 C and 120 C in autoclave (15 psi) was also tested. Finally, the effect of reaction time ranging
163 from 15 to 45 min was evaluated. After hydrolysis, the samples were cooled to room
164 temperature, centrifuged and the supernatant containing reducing sugars was collected and
165 analyzed using the phenolsulfuric acid and dinitrosalicylic acid (DNS) assay. The sugar yield
167 (2)
169 The wet biomass generated after harvesting was used for extraction of lipids and sugars. For
170 combined extraction, the most suitable pretreatment conditions obtained from the previous
171 observations were selected, and thus carried out using bead-beating and acid pretreatments in
172 autoclave. In the process sequence, first algal biomass was treated with the bead-beating for 15
173 min followed by acid treatments at 120 C, 15 psi for 30 min. From the reaction mixture, sugars
174 were obtained from the supernatant whereas lipid was extracted from the residual biomass using
175 chloroform-methanol (2:1) mixture. Lipid was extracted at the endpoint of the pretreatment
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176 reaction and not after the bead-beating, this is to cut down the downtime required for solvent
177 evaporation.
181 Conversion of microalgal lipid to fatty acid methyl esters (FAME) was carried out using acid
182 based transesterification process. The better performing sulfuric acid was used for
183 standardization of the transesterification process. The critical process parameters such as molar
184 methanol to oil ratio (25200), catalyst concentration (0.54.5M), reaction temperature (3080
185 C) and time (39 h) were performed sequentially to obtain the optimal transesterification
186 condition for maximum conversion of algal lipids into FAMEs. The transesterification was
187 carried out in airtight capped glass vials (10cc, Borosil) and the reaction mixture was kept in a
188 temperature controlled incubator. The percentage FAME conversion was calculated using Eq. (3)
191 The suspension remaining after pretreatment was centrifuged, neutralized with NaOH (to nearly
192 pH 6) and the liquor containing sugar fraction was concentrated suitably to test its fermentability
193 using Saccharomyces cerevisiae. Yeast was grown up to mid-log phase in YPD (0.3% yeast
194 extract, 0.3% peptone, and 2% glucose) medium at 30 C, 150 rpm for 48 h. The growth of yeast
195 was monitored at optical density of 600 nm using spectrophotometer. For fermentation
196 experiment, algal hydrolysate was used instead of glucose in YPD medium, inoculated with 5%
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197 (v/v) seed culture of yeast and grown anaerobically at 30 C, 150 rpm for 72 h. Periodic
198 measurements of the concentrations of cells density, ethanol and sugars were carried out. All the
199 experiments were carried out in triplicates and the average value with standard deviation (SD)
202 The total lipids were determined by extracting the algae with chloroform-methanol and was
203 quantified gravimetrically as percent lipid on a dry weight basis.14 The phenolsulfuric acid
204 method was applied to quantify the amount of sugars; the reaction mixture was mixed with 1 mL
205 of 5% (w/v) phenol solution, 1 mL microalgae solution and 2 mL of concentrated sulfuric acid
206 for 1020 min. The absorbance of the characteristic yellow-orange color was measured under the
209 The high performance thin layer chromatography (HPTLC) was used to analyze the
210 transesterified samples at room temperature to calculate the percentage FAME conversion and
211 was also corroborated with gas chromatography (GC). Two microlitre of sample mixed with
212 hexane was loaded onto a TLC silica gel plate (20 x 20 cm, silica gel 60 F254, MERCK,
213 Germany). The mobile phase used was a mixture of hexane, ethyl acetate and acetic acid
214 (90:10:1). The TLC plate was air dried and then scanned through HPTLC scanner 3 (CAMAG,
215 Switzerland) at 208 nm wavelength. Methyl palmitate standard was run in a lane to compare the
216 position of FAME on the plate. The percentage conversion was calculated using Eq. (3).25
217 (3)
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218 where, AMG, ADG, ATG and AFAME correspond to the areas under the peaks of monoacylglycerols
219 (MAG), diacylglycerols (DAG), triacyglycerols (TAG) and FAME respectively, as obtained
220 from HPTLC chromatogram (Fig. S2). Experiments were carried out in triplicates and results
223 The ethanol concentration was analyzed using gas chromatography (GC) (Clarus 500,
224 PerkinElmer) equipped with PLOT-Q capillary column (30 m length and inner diameter of 0.32
225 mm) and flame ion detector (FID). The injector, detector and oven temperatures were maintained
226 at 150 C, 200 C and 50120 C, respectively. Nitrogen gas was used as the carrier gas. The
227 bioethanol concentration was quantified using a calibration curve prepared by injecting different
231 Present investigation highlights the downstream processing steps involved after microalgal
232 biomass cultivation to obtain lipids and carbohydrates for biofuels production. The downstream
233 processing steps were divided into three stages; (i) harvesting of algal culture or slurry through
234 flocculation method to obtain concentrate biomass, (ii) algal biomass pretreatment for the
235 extraction of microalgal lipid and carbohydrate using various methods including cell disruption,
236 solvent extraction and chemical hydrolysis, and finally (iii) conversion of extracted lipids and
237 sugars into biodiesel and bioethanol via transesterification and fermentation, respectively. Fig. 1
238 shows a flow scheme involving different steps carried out in this study. This study also
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239 demonstrated an integrated approach of simultaneous extraction of lipids and carbohydrates from
240 wet biomass for the production of biodiesel and bioethanol in a biorefinery model.
This stage corresponds to the first four boxes to the left in Fig. 1. This study was conducted to
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242
244 ability of Chlorococcum sp. was used as control in order to assess and compare the harvesting
245 efficiency with different flocculants such as ferric chloride (FeCl3 6H2O) and alum (Al2(SO4)3
246 12H2O) (Fig. 2a). For preliminary screening experiment, the initial and final OD reading of
247 culture samples at time zero min and 30 min were noted. It was observed that harvesting
248 efficiency of Chlorococcum sp. culture was significantly influenced by the change in FeCl3
249 concentrations (Fig. 2a). Maximum microalgal flocculation efficiency of 85.93.5% was
250 obtained with the FeCl3 dosage of 100 mg L-1. These results were in good agreement with the
251 reported literature where use of FeCl3 at 200 mg L-1 under low pH as a coagulant was found to
253 Various properties of microalgae that affects their separation from aqueous medium include size,
254 shape, surface charge, specific gravity, motility, growth phase, presence of appendages and
255 extracellular organic matter (EOM) composition and concentration.26 On the other hand, algal
256 harvesting is greatly influenced by the culture medium properties, including cell concentration,
257 pH and ionic strength. Though various mechanisms of flocculation has been extensively
258 explored, flocculation by the use of multivalent metal cations has been reported to be simple and
259 less energy intensive. It was found that such metal ions in the growth medium were hydrolyzed
260 to form positive precipitates, which coagulate negatively charged microalgal cells by sweeping
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261 flocculation and charge neutralization.27 Flocculation can also occur due to change in physical-
262 chemical properties of microalgal cells i.e. their surface charge. However, literature reports also
263 suggests that polysaccharides released by many microalgae species during growth aid in the
264 flocculation by bridging mechanism.27 In the current study, the staining of cells with Alcian blue
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265 dye revealed the binding of EPS to the cells (Fig. S1) which promoted the self-flocculation of
267 suspension and this occurs due to charge neutralization and/or polysaccharides bridging, forms
268 larger aggregates followed by the agglomeration of these into larger flocs that settle to the
270 Next, we explored the influence of the different algal cells densities in the culture medium and
271 found that with the increase in biomass density (0.251.5 g L-1) the harvesting efficiency was
272 also enhanced. Fig. 2b shows time profile with the change in biomass density (0.251.5 gL-1)
273 using FeCl3 (100 mg L-1) at pH7.8, whereas control set was carried out in the absence of FeCl3 at
274 pH7.8. Maximum harvesting efficiency of 90.62.8% was achieved in 30 min of flocculation
275 reaction using 100 mg L-1 FeCl3 with biomass culture density of 1.5 g L-1. The literature reports
276 also suggest that ferric chloride was most suitable flocculant for harvesting, as it showed no
277 deleterious effects on algal growth and least influence on the cell physiological activity and cell
278 components. Most importantly, it is reported that the culture medium can be reprocessed after
279 performing ferric chloride based harvesting, on the contrary, low concentrations (< 5 ppm) of
281 3.3. Biomass pretreatment for the extraction of lipids and carbohydrates
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282 Pretreatment is a succeeding step after dewatering and harvesting of microalgal biomass from
283 culture slurry. Efficient pretreatment of biomass feedstock is often recommended to achieve high
284 biofuel yields. To date pretreatment of biomass is considered one of the most important and
285 expensive step.29 Pretreatment of microalgal biomass involves processes such as cell disruption,
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286 solvent extraction and chemical treatment of microalgal biomass to enable maximum recovery of
288 pretreatment methods on algal biomass was evaluated with respect to extraction yields of lipid
289 and carbohydrate, carried out in separate and simultaneous mode. In Fig. 1, this section
290 corresponds to the middle three boxes. The centered box represents cell disruption, which is also
291 the preliminary step in the pretreatment processes for the recovery of lipids and carbohydrates.
293 Various cell disruption methods were employed to improve the mass transfer and thus increase
294 the extraction efficiency of lipid and carbohydrate. Typically, the lipid content of Chlorococcum
295 sp. was found to be averaging 25% (dcw, w/w) when grown under normal condition, whereas
296 lipid content as high as 45% (dcw, w/w) was obtained under nitrogen limiting condition (data not
297 shown). Cell disruption assisted by physical and chemical means were carried out through bead-
298 beating, homogenization, sonication, lyophilization, acid treatment and autoclaving, and the lipid
299 was extracted using chloroform/methanol (1:2, v/v) as extraction solvents (Fig. 3a). Maximum
300 lipid recovery of 79.53.2% was obtained when the biomass was pretreated with bead-beating.
301 Subsequent maximal lipid recovery of 73.62.5% and 70.43.1% was obtained using
302 autoclaving and homogenization, respectively, although the difference was rather small to bead-
303 beating, whereas cell disruption without any pretreatment as negative control resulted in lipid
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304 recovery of merely 12.11.8% (Fig. 3a). Typically, cell disruption not only improves access to
305 storage lipids but also releases protein and carbohydrates.21 Subsequently, the time duration of
306 bead-beating of algal biomass for lipid recovery was varied from 5 to 25 min and the time length
307 of 15 min was noted as optimal (85.52.4%) (data not shown). While considering the one time
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308 extraction, similar observation has been reported where bead-beating treatment resulted in
310 are reported to be bead milling and high speed homogenization, where the mechanism of solid-
311 liquid interfacial shear forces work that result in high cell disruption efficiency.31
313 Fig. 3b shows the extracted lipid recovery of microalgae using various single and binary solvent
314 systems at room temperature. Among the single solvents system, lipid recovery was recorded to
315 be 52.52.5% (w/w) in chloroform, whereas amidst the binary solvents, chloroform/methanol in
316 2:1 ratio reported maximum lipid recovery of 96.22.9% (w/w). A similar observation has been
317 reported using various microalgal species where chloroform/methanol is best suited for
318 extraction of lipid.32 The lipid extraction with the hexane and hexane/methanol solvents were
319 lower as hexane being non-polar solvent mainly extract neutral lipids.33 Fig. 3b shows the
321 When a microalgal cell is exposed to a non-polar organic solvent, such as hexane and
322 chloroform, the solvent penetrates the cell membrane and interacts with neutral lipids (i.e.,
323 triglycerides) present in the cytoplasm. The solvent interacts with lipids because of van der Walls
324 forces to form an organic solvent-neutral lipid complex. This complex, driven by a concentration
325 gradient, diffuses across the cell membrane and the static organic solvent film surrounding the
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326 cell, enable organic solvent to enter the cells.34 As a result, the neutral lipids are extracted from
327 the cells and remain dissolved in the non-polar organic solvent. Some neutral lipids are found in
328 the cytoplasm as a complex with polar lipids (i.e., phospholipids, glycolipids, etc.). This complex
329 is strongly linked via hydrogen bonds to proteins in the cell membrane. The van der Waals
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330 interactions formed between non-polar organic solvents and neutral lipids are inadequate to
332 ethanol as a polar organic solvent. Mixtures of chloroform/methanol are used to ensure complete
333 extraction of total lipids including the neutral lipids present in the form of free globules and in
334 the form of complex associated proteins. Thus, these mixtures extract both neutral and polar
335 lipids by chloroform and methanol, respectively, and are considered efficient in the extraction of
338 We explored a mild chemical pretreatment method for the extraction and conversion of the algal
339 carbohydrates into fermentable sugars (Fig. 4). In this study, various acid (H2SO4 and H3PO4)
340 and alkali (NaOH and ammonium) chemicals were used for pretreatment of microalgal biomass
341 (Fig. 4a). The sugar recovery was most efficient with NaOH and H2SO4. Thus, in the subsequent
342 experiment, different concentrations of NaOH and H2SO4 were studied with the microalgal
343 biomass (Fig. 4b). The higher sugar recovery in NaOH relative to the H2SO4 could be because
344 NaOH is a known as an effective agent for lysing entire microalgae cells, especially used for
345 protein extraction under mild conditions.11 Not only does it extract carbohydrates embedded both
346 within the cell itself and in the cell wall, but also extracts glycoproteins, contrastingly sulfuric
347 acid treatment tends to primarily extract carbohydrates. We next explored the effect of operating
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348 temperatures and reaction time on microalgal hydrolysis with both acid and alkali pretreatment.
349 The release of sugars was most effective when Chlorococcum sp. was pretreated with sulfuric
350 acid at higher temperature (Fig. 4c). This indicates that an increase in temperature favors the
351 activity of the acid. Typically, there is a trade-off between acid concentration and temperature,
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352 the more dilute acid require higher temperatures, and vice versa, then the treatment with higher
354 quick enough to act upon these complex sugars at elevated temperatures, it is possible that
355 sodium hydroxide acts more slowly in comparison, leading to the direct solubilization of
356 complex microalgal carbohydrates. Such an effect has been seen in the literature regarding
357 sulfuric acid at temperatures above 160 C.10 Literature also indicates that alkaline pretreatments
358 tend to be less effective than acid ones, which could explain the disparity between the two
359 treatments.36 The maximum recovery of 89.63.1% sugar yield was obtained when the biomass
360 was acid pretreated at the optimal condition of 121 C (15 psi) under autoclave in mild acidic
361 condition (0.3N H2SO4) concentration for 30 min (Fig. 4d). Pretreatment of algae by acid
362 hydrolysis is a potentially effective and low cost method.5 The polysaccharides like celluloses
363 and starches are solubilized under acidic conditions, and the combination of high temperature
364 under pressure increases the rate of their hydrolysis into monosaccharides.35 The small portion of
365 algal carbohydrate remained at the endpoint of reaction, suggesting that algal starch might be
367 Carbohydrate content in Chlorococcum sp. represented nearly 24.70.6% (w/w), which
368 comprised mostly of glucose and other component such as mannose, xylose and galactose were
369 also found. Sugars composition examined by HPLC represented peak overlap, this could not rule
370 out that this overlap peaks constituted a heterogeneous mixture of sugars. A region of
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371 overlapping peaks due to sever co-elution may represent some combination of mannose,
372 galactose and xylose.23,37 Xylose and glucose were confirmed separately using GOD-POD assay
373 and Bial's test (orcinol method). Remaining sugars could be substantiated as mannose and
376 In this section, both stepwise and concomitant extraction of carbohydrate and lipid were carried
377 out using the wet biomass (Table 2). Microalgal biomass of Chlorococcum sp. was initially
378 pretreated with the bead-beating followed by autoclaving under acidic condition, then from the
379 reaction mixture the sugars were obtained from the supernatant whereas lipid was extracted from
380 the residual biomass. During the simultaneous extraction process, maximum sugar and lipid
381 recovery of nearly 86.52.6% and 74.11.8%, respectively, was achieved (Table 2). The
382 favorable process condition for extractions were achieved with initial pretreatment with bead-
383 beating for 15 min followed by autoclaving (121 C, 15 psi) in mild acidic condition (0.3N
384 H2SO4) for 30 min. Cell disruption increases mass transfer to support the extraction of both
385 sugars and lipids.38 It is well documented that cell disruption not only releases the storage lipids
386 but also protein and carbohydrates.21 In the concomitant extraction process, the recovery of sugar
387 was found to be excellent after acid pretreatment, however some extraction loss of lipid was
388 observed, which might be due to hydrolysis of lipid or single solvent extraction step (one time) at
389 the endpoint of the process.33 Lipid extracted after hydrolysis is reported to have decreased
390 phospholipids (PL) content, however higher free fatty acids (FFA) content, which could be
391 totally converted to FAME and most of TAG, DAG and MAG were also transformed into
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392 FAME. In addition, the conversion rate of TAGs in lipids was found to be higher than that of
394 Mild treatment with mechanical and non-mechanical methods for short duration could result in
395 optimal results in addition to lower energy consumption. Recent studies suggests that for
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industrial scale mechanical methods are ideal, whereas non-mechanical methods have lower
397 energy needs and may provide uniform cell disruption with higher selectivity.31 Similar
398 observation was reported where efficiency of acid-catalyzed hot-water extraction of lipids from
399 C. vulgaris was found to be around 8590% under a 1% sulfuric acid concentration incubated at
400 120 C for 60 min.33 Temperature has known to be an important parameter in pretreatment of
401 biomass, its effect was tested by varying temperature from 25120 C.39 At high temperatures,
402 cell lyses and simultaneous extraction of metabolites (lipid and carbohydrate) were indeed far
403 more effective, and additional pressure caused by higher temperature in autoclaving than normal
404 boiling point might have a positive effect on disrupting cells as well.39 Moreover the very
405 addition of acid in this process assist especially in hydrolysis of starch while extract lipid
406 simultaneously. The remnant biomass could be effectively used as animal feed or for anaerobic
409 This stage corresponds to the last two boxes to the right in Fig. 1. The final step in downstream
410 processing was the conversion of extracted lipid and carbohydrate after pretreatment into
411 products, biodiesel and bioethanol. Typically, lipids are converted into fatty acid methyl esters
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413 convert triglycerides into esters. The carbohydrates that are hydrolyzed or broken down into
Fig. 5 shows the effect of various process parameter of transesterification reaction for the
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416
418 have high concentration of free fatty acids.32,40 Therefore acid-catalyst based transesterification
419 reactions are applied to algal lipid to produces FAME that can be used as a biodiesel.40 Operating
420 conditions such as solvent to oil ratio, acid concentration, temperature and time, were
421 standardized using Chlorococcum sp. derived lipid. The sulfuric acid identified as suitable
422 catalyst was used for transesterification process. The catalyst amount was varied from 0.54.5M,
423 and showed profound effects on conversion yields, the conversion increased exponentially as the
424 catalyst amount increased up to 3.5M. Higher catalysts concentration increased the contact
425 opportunity of the catalyst and the reactant, which directly influenced the reaction speed and the
426 conversion.41 Similarly, a molar ratio higher than the stoichiometric molar ratio of methanol was
427 required to shift the equilibrium forward toward the formation of FAME. Temperature clearly
428 influenced the FAME conversion but remained constant beyond 70 C. Maximum FAME
429 conversion yield of 95.22.8% was achieved, with the operating reaction condition of 100 molar
430 ratio of methanol to oil, 3.5M acid concentration, conducted at 70 C for reaction time of 7 h
431 (Fig. 5) (Fig. S2d). More than 90% of the conversion occurred in 5 h of incubation time, and
432 after 7 h the conversion remained nearly constant at almost 95% because of a near equilibrium
433 conversion.41 These results also indicate that saponifiable lipids other than triglycerides, such as
434 PL and glycolipids are transformed into FAMEs by this method. Moreover lipid extracted after
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435 hydrolysis is reported to have decreased PL content, and most of TAG, DAG, MAG and FFA are
436 also transformed into FAME with high conversion rates of nearly 98% via transesterification.8
437 The results were corroborated with the literature where 4.5M HCl was favorable for FAME
438 conversion.32
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440 After acid pretreatment, the fermentability of microalgal sugars to bioethanol using S. cerevisiae
441 was investigated, the initial sugar concentration of 20 g L-1 was used. Fig. 6 showed the time
442 course profile of ethanol concentrations and residual sugar during the bioethanol fermentation
443 process. In the fermentations process, almost 90% of sugar was consumed within 72 h.
445 the cells and/or assimilation of a secondary substrate.37 Ethanol concentrations peaked at 48 h
446 and stayed almost constant by 72 h. The maximum ethanol yield of 4.10.25 g L-1 was observed.
447 The ethanol yield percentage was 40% of the theoretical yield, this yield could be increased by
448 using competent strain of S. cerevisiae. The result obtained compare favorably with published
449 data where ethanol yield ranged from 2 to 5.6 g L-1 by S. cerevisiae in the fermentation of
452 Techno-economic analyses (TEA) of various algal biofuel processes have been reported.
453 However literature reports have shown large variations in the calculated fuel cost with 30%
454 accuracy.43 Depending on the process design and raw material inputs, the biofuel cost was
455 reported to vary from a low value of $1.65 gal-1 to a higher value of $33.16 gal-1.44,45 Significant
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456 reduction in variability within a harmonized range from $11.68 $14.31 gal-1 was achieved
457 through meta-analysis.46 When TEA was carried out based on comprehensive mass balances, the
458 cost of biodiesel production was determined to be $9.84 gal-1 for open algal ponds and $20.53
459 gal-1 for closed photobioreactors.47 Another report on detailed techno-economic analysis of
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460 optimized production processes indicated improved cost estimations with the final costs of
462 Though technical feasibility of microalgal biodiesel has been established, economic viability will
463 ultimately depend on new technological breakthroughs and of course, on the cost of crude oil.48
464 An integrated process in a biorefinery model that generates more than one value-added product
465 from microalgal biomass feedstock can considerably reduce the cost of the overall process and
466 make the process profitable and feasible.49 However, there are hardly any reports on techno-
467 economic analysis of biofuel production in a biorefinery. Harun et al. reported substantial
468 reduction in the cost of biodiesel production by nearly 33% in a biorefinery producing both
469 biodiesel and methane.48 A similar study, wherein the carbohydrate and lipid based combined
470 biofuel yields showed cost cutting by up to 33% when compared to a lipids-only process, proved
472 4. Conclusions
473 This study convincingly demonstrated an integrated approach in downstream processing for
474 concomitant extraction of lipids and carbohydrates for the development of microalgal based
475 biofuel biorefinery to obtain both biodiesel and bioethanol as potential liquid fuels. A series of
476 harvesting and pretreatment experiments confirms the feasibility of the key process steps in the
477 proposed holistic approach. This experimental work confirmed the ability to use wet algal
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478 biomass for pretreatment to achieve encouraging biofuel yields. The process resolves the current
479 major limitations to the economic and energetic feasibility of algal biofuels by integrating some
480 of the unit operations involved in pretreatment of algal biomass for simultaneous extraction of
481 lipids and sugars. Thus, this holistic approach is expected to provide operational flexibility and
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482 feasibility by integrating some of the steps in downstream processes for valorizing microalgal
484 smokestacks, where they can digest the pollutants and deliver the feedstock for biofuels. The
485 present investigation presented the potential of operational simplicity in downstream processing
486 especially from harvesting of algal cells to extraction of potential microalgal metabolites for their
487 conversion into biofuels to promote commercial scale production. This synergetic approach of
488 biofuels production could offer a sustainable alternative to current methods in catering the
489 energy demand and replacing petroleum-based fuels for sustainable development of bio-based
491 Acknowledgements
492 The authors gratefully acknowledges Department of Biotechnology (DBT), Govt. of India for
493 fellowship, IIT Kharagpur, and Department of Science & Technology (DST), Govt. of India, for
494 the financial support for the project (No.: DST/IS-STAC/CO2-SR-160/13(G); Date: 08.07.2013).
495 AK thankfully acknowledges Prabuddha Dey, Atrayee Chattopadhyay and Shanti Kiran for their
497 References
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508 173.
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591
592 Tables
593
51.47
H, % 7.91
596
597
598
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603
605 Fig. 1 Process scheme of combined biodiesel and bioethanol production using microalgal
606 culture.
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607 Fig. 2 Harvesting efficiencies of Chlorococcum sp. using (a) alum and FeCl3 at varying
610 Fig. 3 Lipid extraction from Chlorococcum sp. biomass (a) effects of various cell disruption
611 methods (b) effect of various single and binary solvent systems on lipid recovery from biomass.
612 Fig. 4 The yield of sugar release under different conditions of pre-treatment (a) effect of various
613 chemical pretreatment (b) effect of chemical concentration (c) effect of temperature and (d)
614 effect of reaction time.
615 Fig. 5 Effect of various critical parameters on conversion of algal lipid into FAME using
616 transesterification process; a) molar ratio of methanol to oil b) acid concentration c) temperature
617 and d) reaction time.
618 Fig. 6 Time course profiles of bioethanol production and total sugar depletion via fermentation
619 of algal sugar by S. cerevisiae.
620
621
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