Chakravarthy Kavitha, Veeramuthu Ashokkumar, Senthil Chinnasamy, Sailendra Bhaskar, and Ramasamy Rengasamy, Pretreatment of Lipid extracted

Botryococcus braunii Spent Biomass for Bio ethanol Production, Int.J.Curr.Biotechnol., 2014, 2(1):11-18.

International Journal of Current

Biotechnology
ISSN: 2321 - 8371
Journal Homepage : http://ijcb.mainspringer.com

Pretreatment of Lipid extracted Botryococcus braunii Spent Biomass for Bio

ethanol Production
Chakravarthy Kavitha*1, Veeramuthu Ashokkumar1, Senthil Chinnasamy2 , Sailendra Bhaskar2 ,
and Ramasamy Rengasamy1
1
Centre for Advanced Studies in Botany, University of Madras, Guindy campus, Chennai-600025, Tamilnadu, India.
2
Aban Infrastructure Private Limited (Biotechnology Division), Chennai 60008, Tamil Nadu, India.

A R T I C L E I N F O A B S T R A C T

The microalgae Botryococcus braunii has gained more attention among biofuel
Article History: industries due to its ability to produce high amount of lipids and carbohydrates. The
Received 08 January 2014 aim of the present study is mainly focused on bioethanol production from the spent
Received in revised form 10 January 2014 biomass of Botryococcus braunii. The lipid extracted alga or spent biomass was
Accepted 20 January 2014 subjected to different acid pretreatments using Sulphuric, Hydrochloric, Oxalic,
Available online 25 January 2014 Fumaric, Malic, Orthophosphoric, and Nitric acids and alkali pretreaments using
NaOH, KOH, CaOH, and Liquid ammonia. The efficiency of treatments was assessed
in terms of release of fermentable sugars from the spent biomass to increase bioethanol
yield. All the fermentation studies were conducted using the baker ’s yeast
Key words: Saccharomyces cerevisiae. Among all the acid and alkali pretreatments, Ammonia
Bioethanol; Botryococcus braunii; fiber explosion (AFEX) treatment showed maximum recovery of sugars (0.5 g g-1 of
Fermentation; Hydrolysis; Pre biomass) and yield of ethanol (0.232 g of ethanol g-1 of biomass). However for
treatments; Spent biomass.
commercial application ammonia has to be recovered in order to make the pretreatment
process cost effective.
generation biofuels such as lignocellulosic ethanol show
Introduction great promise, lots of technologic challenges need to be
Microalgal biomass can serve as a potential source of addressed to commercialize bioethanol production from
biofuel feedstock to meet the present and future energy ligncellulosic residues.Lignocellulosic biomass is difficult
and fuel needs. The two most commonly used biofuels to hydrolyze as it needs to be converted into six carbon
are biodiesel and bioethanol which are blended with the sugars for bioethanol production. The enzymes used for
conventional fossil derived fuels such as diesel and saccharification are costly and the removal of lignin from
petrol/gasoline, respectively (John et al., 2011) lignocellulosic biomass is considered challenging.
Bioethanol is considered as an environmental friendly Considering the above, third generation biofuel feedstock
alternative fuel source due to its low toxicity, such as algae which accumulate carbohydrates as starch
biodegradability, and its ability to effectively blend with can be used for bioethanol production. The starch and
petrol/gasoline without any engine modification (Harun cellulose present in the algal biomass are made of long
et al., 2010). Bioethanol blended petrol/gasoline has chain of glucose molecules. Hence, the pretreatment of
the potential to solve the global energy problem in future the algal biomass which contains no lignin is a simple
in view of increasing demand for fossil crude which process and it is considered very cost effective.
results in environmental pollution due to green house
gas emissions (Guragain et al., 2011). Microorganisms such as bacteria and yeasts have the
capability to ferment simple and complex sugars for the
Microalgae can be utilized for bioethanol and biodiesel production of bioethanol. Saccharomyces cerevisiae is
production as they are rich in carbohydrates and lipids the most commonly used yeast for bioethanol production
(Table 1). When compared to other bioethanol sources, in different industries. The yeast S. cerevisiae can
microalgae require unproductive/waste lands and poor produce bioethanol up to 18% of the fermentation broth.
quality waters such as seawater, brackish water and It has the ability to tolerate a wide range of temperature,
wastewaters which include sewage, agricultural and inhibitors and osmotic pressure (Banerjee et al., 2010).
industrial effluents for cultivation. Though second
*Corresponding author.
Hydrolysis of carbohydrates can be achieved by breaking
Email address: auroaparna@gmail.com.
down the complex sugars present in the biomass such as
Mobile no: +918754886646.
cellulose and hemicellulose to release fermentable sugars
Tel/Fax: +914422353309/22352494.
(Mosier et al., 2005a). The hydrolysis of cellulose was

11 Int.J.Curr.Biotechnol. Volume 2; Issue 1; Jan, 2014

achieved by physical cracking followed by a chemical It was cultivated in 1500 L capacity open race way pond
catalytic process at high temperature and pressure using Modified CFTRI medium (Urea -0.4 g-L-1, DAP -
(Mcmillan, 1994). The commonly used methods include 0.016 gL-1, Potash - 0.234 gL-1, NaHCO3 – 1 gL-1, MgSO4 -
milling and grinding, steam explosion and ammonia fibre 0.5 gL-1) at pH 7.5 for a period of 15 days. Biomass was
explosion. Acid pretreatment results in solubilizing the harvested on 15th day, shade dried for 3 days, pulverized
hemicellulose of the biomass making the cellulose more using mixer- grinder and estimated for total carbohydrates
accessible for the enzymes. This type of pretreatment (Dubois et al., 1956), proteins (Bradford, 1976) and lipids
can be performed with the concentrated or dilute acids, (Folch et al., 1957). A known quantity (1 kg) of dry
but the utilization of concentrated acid resulted in biomass was taken and extracted for total lipid using
reduction of ethanol production due to the formation of Chloroform: Methanol 2:1 in Soxhlet apparatus. The spent
inhibitory compounds (Wyman, 1996). algal biomass after lipid extraction was used for
bioethanol production.
Higher bioethanol yields in hydrolysis have been
recorded when pretreating the lignocellulosic biomass Pretreatment Methods
with dilute sulphuric acid. The use of hydrochloric, The spent algal biomass of B. braunii was subjected to
phosphoric and nitric acids in the pretreatment of biomassacid and alkali pretreatments to produce sugar
was reported in earlier studies (Mosier et al., 2005a). Alkali
hydrolysates for fermentation. Different acids such as
pretreatment is effective depending on the lignin content Sulphuric, Hydrochloric, Oxalic, Fumaric, Malic,
of the biomass. It increases the cellulose digestibility Orthophosphoric and Nitric acids and different alkalis
and enhances solubilization of lignins. Alkali pretreatment
such as Sodium hydroxide (NaOH), Potassium hydroxide
can be performed even at room temperature which results (KOH), Calcium hydroxide (CaOH), and Liquid ammonia
in reduced sugar degradation than acid pretreatment (NH3 (L)) were used at different concentrations viz., 0.5,
(Carvalheiro et al., 2008; Kumar et al., 2009). The present
1.0, 2.0, 3.0, 4.0 and 5.0% (v/w). For the pretreatment
investigation aims at assessing the potential of various experiments, 25 g of spent biomass was dissolved in 100
acid and alkali pretreatment techniques on the yield and ml of distilled water containing different concentrations
recovery of sugars and bioethanol from the biomass of of acid and alkali agents. This mixture was autoclaved at
lipid extracted alga Botryococcus braunii. 121oC at 15 psi for 40 min to hydrolyze the biomass. The
hydrolysates obtained after the pretreatment process was
Materials and Methods centrifuged at 10000 rpm for 10 min to separate the sugar
Microalgal biomass solution. The sugar content in the supernatant was
The green alga, Botryococcus braunii AP102 (Accession estimated using the method suggested by Dubois et al.
No:JQ585724) was obtained from the Algal Culture (1956). The supernatant was then neutralized to pH 7
Collection Facility, Centre for Advanced Studies (CAS) and it was autoclaved again at 121oC at 15 psi prior to
in Botany (CAS), University of Madras, Chennai, India. fermentation.
Table 1: Chemical composition of different microalgal strains (% of dry matter) (Becker, 2007)
Algal S trains Carbohydrates Lip ids Proteins
(dry wt%)
Anabaena cylindrica 25-30 4-7 43-56
Aphanizomenon flos-aquae 23 3 62
C hlamydomonas rheinhardii 17 21 48
C hlorella pyrenoidosa 26 2 57
C hlorella vulgaris 12–17 14–22 51–58
Dunaliella sali na 32 6 57
Euglena gracil is 14–18 14–20 39–61
Porphyridium cruentum 40–57 9–14 28–40
Scenedesmus obliquus 10–17 12–14 50–56
Spirogyrasp. 33–64 11–21 6–20
Arthrospira maxima 13–16 6–7 60–71
Spirulina platensis 8–14 4–9 46–63
Synechococ cussp. 15 11 63
Botryococcus braunii AP102 (After
47.2 4.2 20.5
lipid extraction)a
a
present study
Table 2: Pretreatment of spent algal biomass with strong acids

Glucose yield Bioethanol yield

-1 -1
Concentration (g g of (g g of Bioethanol yield
Pretreatment
(%) spent algal spent algal (Wt. %)
biomass) biomass)
Sulphuric acid 0.5 0.294 0.142 14
H ydrochloric acid 0.5 0.251 0.131 13
Nitric acid 5.0 0.209 0.104 10

Volume 2; Issue 1; Jan, 2014 Int.J.Curr.Biotechnol. 12

 Table 3: Pretreatment of spent algal biomass with organic acids
Glucose yield Bioethanol yield
-1
Concentration (g g of (g g -1 of Bioethanol yield
Pretreatment
(%) spent algal spent algal (Wt. %)
biomass) biomass)
Fumaric acid 0.5 0.174 0.087 9
Oxalic acid 0.5 0.110 0.056 6
Malic acid 4.0 0.104 0.050 5
Orthophosphoric acid 3.0 0.055 0.027 3

Table 4: Pretreatment of spent algal biomass with alkali agents

Glucose yield Bioethanol yield

-1 -1 Bioethanol
Concentration (g g of (g g of
Pretreatment yield
(%) spent algal spent algal
(Wt. %)
biomass) biomass)
AFEX 3.0 0.500 0.232 23
KOH 4.0 0.360 0.180 18
NaOH 4.0 0.350 0.168 17
CaOH 1.0 0.076 0.036 3

Microalgal Fermentation methodology for the release of sugars from B. braunii

Baker’s yeast “Saccharomyces cerevisiae” was obtained
from the local market, Chennai, India for this study. The
yeast was cultured in Luria Broth (LB) medium maintained
at pH 4.8. A 3% (v/v) inoculum of yeast was added to 100
ml of sterilized LB medium and kept in a shaker at 200 rpm
at 30ºC for 24 h. Then the yeast cells were centrifuged at
5000 rpm for 10 min and harvested. The supernatant was
discarded and the cells were washed with 1% (v/v) of
orthophosphoric acid as per the procedure described by
Harun and Danquah (2011) for three times in order to
remove residual sugars. Yeast inoculum (3% v/v) was
added into the airtight bottles containing 50 ml (v/v) of
algal hydrolysates and incubated in dark at 200 rpm in a
rotary shaker for 4 days. The bioethanol produced after
fermentation was distilled at 78.3ºC for 30 min in an
ethanol distillation unit and quantified.
Estimation of ethanol
The concentration of ethanol was estimated by adopting
the method described by Caputi etal. (1968) with minor
modification. One ml of chromic acid solution (0.5g of
potassium dichromate dissolved in 5ml of distilled water
and 45 ml of sulphuric acid) was added to one ml of the
sample and kept in a water bath at 800C for 10 min. It was
then cooled and the absorbance was measured at 600
nm. Ethanol concentration was measured using a
standard curve prepared by using different
concentrations (1-10% v/v) of ethanol as a standard.
Results and Discussion
Microalgae consist of different types of carbohydrates
which are mostly stored in the cell wall (Kurano et al.,
1998). In order to use the carbohydrate source of
microalgae for fermentation it is necessary to disrupt the
cell wall material. This study uses the acids and alkali
agents in order to identify the best treatment
cells. The Table 5 shows the bioethanol yield from
different microalgal species using various pretreatments.
Microalgal composition
The composition of Botryococcus braunii biomass after
lipid extraction was shown in Table 1. Total carbohydrates
estimated in the B. braunii were 47.2% (w/w) which is
comparable with the carbohydrate content of
Porphyridium cruentum (40-57%) (Becker, 2007). The
carbohydrate content may vary from species to species
and the optimization of pretreatment is necessary to
extract the maximum carbohydrate from the biomass.
Acid pretreatment
Dilute acid hydrolysis has the advantage since it is faster
than other types of hydrolysis including enzymes.
However acidic condition often leads to the degradation
of glucose (Harun et al., 2010). In the present study,
among the seven different acids used 0.5 % sulphuric
acid (H2SO4) pretreated B. braunii biomass showed
maximum glucose and bioethanol yield of 0.294 g and
0.142 g per gram of spent biomass, respectively (Table 2).
Higher concentration of sulphuric acid at 3%, recorded
lowest yields of glucose (0.240 g per g) and bioethanol (
0.119 g g-1per g) of spent biomass. Tutt et al. (2012a)
achieved maximum glucose conversion of 0.276 g g-1in
1% H2SO4 pretreated unrinsed wheat straw and maximum
bioethanol yield of 0.092 g/g from rinsed wheat straw.
Harun and Danquah (2011) reported maximum bioethanol
yield of 7.2 gL-1 from 15 gL-1of microalgae which was
treated using 1% H2SO4 for 25 min at 1400C.
Botryococcus braunii spent biomass when pretreated
with HCl showed maximum glucose and ethanol yields of
0.251 g/g and 0.131 g/g, respectively at 0.5% (v/v)
concentration similar to H2SO4 pretreatment (Table 2).

13 Int.J.Curr.Biotechnol. Volume 2; Issue 1; Jan, 2014

 Table 5: Comparision of bioethanol yield from different microalgal sources.
Microbes used Bioethanol yield
Biomass Pretreatment Conditions References
for fermentation (g g-1 biomass)
Arthrospira platensis (0.5N) Nitric acid S. cerevisiae 0.163 (Markou et
100°C for 180 min. al., 2013)
(0.5N) Sulphuric acid,100°C for 180 0.162
min

Chlorococcum humicola 1% Sulphuric acid, 140°C for 30 min. S. cerevisiae 0.520 (Harun and
Danquah,
2011)

Chlorococcum infusionum 0.75% Sodium hydroxide, 120°C for S. cerevisiae 0.260 (Harun et
30 min. al., 2010)

Chlorella vulgaris 1% Sulphuric acid, 121°C for 20 min. Z. mobilis 0.233 (Ho et al.,
2013a)

Chlorella biomass 2% Hydrochloric acid, 2.5% MgCl2, S. cerevisiae 0.470 g g-1 (Zhou et
180°C for 10 min. glucose al., 2011)

Chlamydomonas reinhardtii 0.2% glucoamylase, 55°C for 30 min. S. cerevisiae 0.235 (Choi et al.,
UTEX 90 2010)

C. reinhardtii UTEX 90 3% sulphuric acid, 110°C for 30 min. S. cerevisiae 0.292 (Nguyen et
al., 2009)

Dunaliella tertiolecta AMG 300L- S. cerevisiae 0.140 (Lee et al.,

0.4ml g-1 of amyloglucosidase, 55°C 2013)
for 12h.

Scenedesmus obliqious CNW-N 2.5% Sulphuric acid, 121°C for 20 Z. mobilis 0.202 (Ho et al.,
min. 2013b)

Scenedesmus abundans PKUAC 3% Sulphuric acid, 110°C, for 30 min. S. cerevisiae 0.103 (Guo et al.,
12 250 mg of Cellulase, 37°C for 30 min. 2013)

-1
Schizocytrium sp. Hydrothermal treatment, 115.5°C for E. coli KO11 11.8 g L of (Kim et al.,
46.7 min. amylase, glucoamylase. ethanol from 25.7 2012)
g L-1 of glucose

Table 6: Bioethanol yield from various feedstock sources in comparison with the estimated yield of bioethanol
from microalga B. braunii used in this study (Sanchez, 2008).
S.No Crop Annual yield Conversion rate Bioethanol yield
(t ha-1) to bioethanol (L ha-1)
(L t-1 )
a
1 Sugarcane (Brazil) 70-88 70 4900- 6160
b b
2 Sugarcane (India) 70-88 10.12 708–900
3 Corn/Maize 5-10 410 2050 – 4100
4 Cassava 40 150 6000
5 Sweet sorghum 35 80 2800
6 Wheat 4 390 1560
c d
7 Microalgae (B.braunii) 30-60 294 8820-17460
a
bioethanol yield calculated per ton of sugarcane juice as bioethanol in Brazil is directly produced from sugarcane juice
a
bioethanol yield calculated per ton of molasses as bioethanol in India is directly produced from molasses yield cassumption ~10
to 20 g m-2 d-1 for 300 days
d
conversion rate to bioethanol calculated based on the results obtained in the study for 3% ammonia solution treatment i.e.0.232
g g-1
Volume 2; Issue 1; Jan, 2014 Int.J.Curr.Biotechnol. 14
The lowest level of glucose and bioethanol yields of 0.209
g and 0.104 g per g of spent biomass were recorded for
the treatment with 3% HCl. Satyanagalakshmi et al. (2011)
achieved a maximum yield of 0.277 g of glucose per g of
water hyacinth treated with 2% HCl. Tutt et al. (2012a)
achieved 0.221g of glucose yield and 0.067g of bioethanol
yield per g of rinsed wheat straw treated with 1% of HCl.
Among different concentrations of both the acids,
maximum glucose and bioethanol yield were recorded at
0.5% concentration which was the lowest concentration
used in the experiments. The concentrations above 0.5%
decreased the level of glucose and bioethanol yield of B.
braunii due to the formation of inhibitors.
The spent biomass of B. braunii when pretreated with
3% ortho phosphoric acid recorded maximum yield of
glucose 0.055 g g-1 of spent biomass and bioethanol 0.027
g g-1 of spent biomass (Table 3). But Vasconcelos et al.
(2013) achieved 98% solubilization of hemicelluloses in
sugarcane bagasse in the treatment carried out with 0.2%
of orthophosphoric acid.
The spent algal biomass when subjected to 4% malic
acid pretreatment resulted in maximum glucose yield of
0.104 g and the bioethanol yield of 0.05 g g-1 (Table 3).
Kootstra et al. (2009) achieved 96% of glucose yield in
50mM malic acid pretreated wheat straw. They also
suggested that malic acid and fumaric acid are the good
alternatives for sulphuric acid as they are very efficient
in the hydrolysis of polysaccharides. Unlike sulphuric
acid, organic acids do not degrade the free sugars to
Furfural and Hydroxy Methyl Furfural.
The spent biomass when pretreated with 0.5% fumaric
acid showed maximum glucose yield of 0.174 g and
bioethanol yield of 0.087 g g-1 (Table 3). Harmsan et al.
(2010) reported the biomass treated with fumaric acid was
very efficient in removing hemicellulosic sugars and it is
specifically suitable for the biomass with low lignin
content. Partanen and Mroz (1999) and Radecki et al.
(1988) reported that the use of organic acids instead of
H2SO4 in the pretreatment significantly improves the
quality of the by-product stream, so that bioethanol
extracted biomass can be used as fertilizer or animal feed
supplement.
Dicarboxylic acids such as oxalic acid are very expensive.
However, this acid can be a potential alternative to H2SO4
for the pretreatment of cellulosic biomass (Kootstra et
al., 2009; Lee et al., 2009; Lu and Mosier, 2007). The
spent algal biomass treated with oxalic acid at 0.5%
showed maximum glucose yield of 0.110 g and the
bioethanol yield of 0.056 g g-1(Table 3). Zhang et al.
(2013) achieved 87.7% of glucose recovery in 0.5% of
oxalic acid pretreated dry maple wood which is very high
with the results obtained with oxalic acid pretreatment in
the present study. Oxalic acid can directly catalyze the
cellulose and hemicellulose hydrolysis. Oxalic acid is
reported as one of the strongest organic acids and it is
less toxic to yeasts and other microbes when compared
to other acids as it lowers the pKa and restricts diffusion
of the ionized forms across the cellular membranes. It
does not inhibit glycolysis and produce noxious odors
when compared to H2SO4 (Mosier et al., 2002).
Nitric acid pretreatment of biomass showed good results
due to (HNO) affinity reaction to break down cellulose
into glucose (Horst et al., 2011). In general, nitric acid
removes most of the hemicelluloses from the biomass
and leaves the cellulose fibres easily accessible for
15 Int.J.Curr.Biotechnol.
enzymes (Tutt et al., 2012a). Among the different
concentrations tested, the treatment with 5% nitric acid
showed better results compared to other concentrations.
Maximum glucose yield of 0.209 g and bioethanol yield
of 0.104 g per gram of spent biomass were recorded at
5% nitric acid pretreatment. There was a gradual increase
in glucose and ethanol yield with increasing concentration
of nitric acid up to 5%. The concentration above 5%
treatment resulted in over foaming of biomass due to
higher acid content. Thus in the present study the
concentration was restricted only up to 5%.The results
were shown in Table 2. Chong et al. (2004) obtained 2.87
g L-1 of glucose using nitric acid pretreatment from
sugarcane bagasse. Tutt et al. (2012a) achieved maximum
glucose conversion of 316.7 g kg-1 and bioethanol yield
of 95 g kg-1 in 1% nitric acid pretreatment of wheat straw.
They also achieved maximum glucose conversion of 324
g kg-1 and bioethanol yield of 96.6 g kg-1 in nitric acid
pretreatment on rye straw (Tutt et al., 2012b).
Alkali pretreatment
In alkaline pretreatment salvation and saponification
reaction takes place which removes the cross links
between the hemicelluloses and other components (Sun
and Cheng, 2002; Hendriks and Zeeman, 2009). It
increases the porosity and decreases the crystallinity of
the cellulose (Galbe and Zacchi, 2007). This process is
more suitable for the materials which contain little lignin
(Harun et al., 2010). In this study four different alkali
agents viz. NaOH, KOH, CaOH and liquid ammonia were
used at different concentrations and the pretreament
efficiency was evaluated in terms of glucose and
bioethanol yield.
The algal biomass treated with 4% KOH and NaOH, gave
comparable glucose yields of 0.360 g and 0.350 g g-1, and
bioethanol yields of 0.18 g and 0.17 g g-1, respectively.
Harun et al. (2010) reported maximum glucose yield of
0.350 g g-1 of microalgal biomass and bioethanol yield of
26.1 wt% in the pretreatment carried out with 0.75% (w/
v) NaOH at 1200C for 30 min. The maximum glucose yield
recorded at 4% NaOH pretreatment in this study was
similar to the observation made by Harun et al. (2010).
Tutt et al. (2012a) achieved maximum glucose yield of
0.268 g g-1 and bioethanol yield of 0.104 g g-1 in 1% KOH
pretreated rinsed wheat straw. They also achieved
maximum glucose yield of 0.245 g/g and bioethanol yield
of 0.080 g/g in 1% KOH pretreated rinsed rye straw (Tutt
et al., 2012b). The result in the present study was higher
than the results obtained by Tutt et al in KOH
pretreatment.
Calcium hydroxide also called ‘slake lime’ is an effective
agent which removes the amorphous substances and
increases the crystallinity index (Nagwani, 1992; Kim and
Holtzapple, 2006). It requires low cost and less safety
measurements when compared to NaOH or KOH and can
be recovered easily from the hydrolysates by reacting
with CO2 (Mosier et al., 2005b). In lime pretreatment,
maximum glucose conversion of 0.076 g and bioethanol
yield of 0.036 g was recorded at 1% CaOH. Rabelo et al.
(2009) acheived maximum glucose yield of 218 mg g-1 in
the pretreatment where 0.040 g of lime was added per g of
sugarcane bagasse which was higher than in the present
study. Thus the lime pretreatment in this study shows
poor hydrolysis.
Ammonia fiber explosion, or AFEX, is an alternative
pretreatment method that has many advantages over
dilute acid hydrolysis. The AFEX treatment removes the
crystallization of the cellulose, partially depolymerises
Volume 2; Issue 1; Jan, 2014
the hemicellulose and cleaves the bond between lignin
and cellulose thereby freeing the cellulose for
degradation (Teramoto et al., 2008). When used as a
pretreatment for enzymatic hydrolysis, and in biomass
with low lignin content, it can result in a cellulose and
hemicellulose conversion of over 90% (Sun and Cheng,
2002). Another advantage of AFEX is that, it does not
produce inhibitory by-products such as Hydroxy Methyl
Furfural and Furfural when compared to other
pretreatments. Yang and Wyman (2008) reported that
there is no need to neutralize the AFEX treated biomass
before enzymatic hydrolysis. It greatly enhances the
digestibility of biomass and even at very low enzyme
levels high glucose and xylose yields are achievable
(Teymouri et al., 2005). Solaiman et al. (1979) reported
that during AFEX pretreatment, there was no sugar loss
and total material mass was increased by 1–2%, due to
the ammonia binding onto the biomass.
In this study, the treatment with 3% liquid ammonia
showed better yields of glucose (0.5 g g-1) and bioethanol
(0.232 g g-1). Garlock et al. (2009) reported that a maximum
glucose yield of 354.4 g kg-1 was achieved in 1.5 g of
ammonia treated corn stover. The above study revealed
that among all the pretreatments of both acid and alkali
the liquid ammonia pretreated spent algal biomass of B.
braunii gave maximum yield of sugars and bioethanol.
Results of the above alkali pretreatments in this study
are summarized in Table 4.
Sugarcane and corn are considered to be the most
productive sources of bioethanol. Brazil is known for
bioethanol from sugarcane and the average annual
bioethanol yield from sugarcane is ~6100 L ha -1 if
sugarcane juice is directly used for fermentation (Table
6). However, in India, bioethanol is mostly produced
from sugarcane molasses which is a waste by-product
obtained after the removal of sucrose from the sugarcane
juice for sugar production. Average bioethanol yield
obtained in India through molasses route is ~700-900 L
ha-1. Based on the results obtained through the 3% liquid
ammonia treatment in the present study, it is estimated
that a maximum of 17000 L ha-1 of bioethanol can be
produced through algal cultivation assuming a
productivity of 20 g m-2 d-1 (Table 6).
Fig 1 shows the conceptual process for the production
of bioethanol and other biofuels from algae to improve
the commercial viability of this technology. Biomass
residues obtained after pretreatment and hydrolysis can
be subjected to anaerobic digestion processs to produce
biomethane and the outlet of the anaerobic digester can
be treated through hydrothermal liquefaction process to
produce green crude which can be further converted to
green diesel, green petrol/gasoline and green jet fuels in
a petrocrude refinery.
Conclusions
In order to increase the bioethanol yield from the
microalgae Botryococcus braunii, different methods of
pretreatments with acid and alkali agents were carried
out to assess their potential in extracting sugars from
the spent algal biomass for further conversion into
bioethanol through fermentation using the baker’s yeast
S. cerevisiae. The results indicated that the AFEX
pretreatment showed maximum yield of bioethanol when
compared to other pretreatments. However, for
commercial applications, the cost economics of this
pretreatment technology should be further improved
through recovery and reuse of ammonia.
Volume 2; Issue 1; Jan, 2014 Int.J.Curr.Biotechnol.
Acknowledgements
The authors are thankful to the management of Aban
Infrastructure Pvt., Ltd., Chennai for the financial support
provided for this study.
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