Results and discussion
Total sugar and reducing sugar (after dilute acid hydrolysis)
were estimated for both E. intestinalis and U.
lactuca. The optimization variables considered are acid
concentration, reaction temperature, substrate concentration and
reaction time. Effect of dilute acid
concentration
The effect of dilute acid concentration on E.
intestinalis and U. lactuca was determined using
acid concentrations of 0.01, 0.05, 0.3, 0.5, 0.7, and 1 N, for
1% substrate concentration at 121oC for 1 hour.
Higher total sugar for E. intestinalis and U.
lactuca biomass was obtained for pretreatment using
H2SO4 (Fig 1.). The sugar content
gradually decreased with increase in acid concentrations as
sugar decomposition varies based on concentrations and different
acid catalytic activities [50]. Acid-catalyzed glucose
decomposition is more dependent on the concentration of hydrogen
ions at a particular temperature than on hydrogen ion sources
[51].
Reducing sugar estimation was carried out for E.
intestinalis and U. lactuca using HCl and
H2SO4 (Fig 2). Reducing sugar increased
gradually with the increase in H2SO4 concentration
for E. intestinalis, whereas for U. lactuca
reducing sugar increased up to 0.5N H2SO4
and then decreased drastically. Acid hydrolysis efficiency was
calculated for both the acid, and it was found that 0.7 N H2SO4
with the conversion efficiency of 80.18% was suitable for E.
intestinalis and 0.5 N H2SO4
achieved a conversion efficiency of 60.07% for U.
lactuca. These concentrations were kept constant for
further optimization study. H2SO4
hydrolysis exhibited better reducing sugars compared to HCl and
was considered for optimization. Hydrolysis using different acid
concentrations released different concentrations of reducing
sugars from the macroalgal biomass depending on their structure
and biochemical composition, demonstrating that a customized
approach is needed for hydrolysis.
Effect of reaction temperature on acid hydrolysis
Reducing sugar at different temperatures of 30, 60, 90, and 120
oC was recorded (Fig 3). The highest reducing sugar
of 549.45 mg g-1 and 528.46 mg g-1 was
obtained for E. intestinalis and U. lactuca at
120 oC with 90.9% and 97.7% sugar conversion,
respectively. Pretreatment of terrestrial biomass involves
higher temperature (165-210oC), this is attributed to
their rigid structures [39], whereas macroalgal biomass requires
milder temperatures. Studies involving Red algae Kappaphycus
alvarezii pretreated using 1%
H2SO4 at 120 oC for 60 min and
obtained 81 g L-1 of reducing sugar [28] whereas,
earlier study [30] of similar pretreatment conditions at 130
oC obtained 22.4 g L-1 of reducing sugar.
Reducing sugar of 65 mg g-1 was obtained after
pretreatment of U.pinnatifida at 120 oC for
24 h [52]. Pretreatment of Saccharina japonica using 40
mM H2SO4 at 121oC for 60 min
yielded 20.6 g L-1 of reducing sugar [37]. An earlier
study [39] of similar pretreatment conditions using 1mM H2SO4
for 120 min achieved 34 g L-1 reducing sugar, indicating that
the concentration of acid in hydrolysis plays a critical role in
incubation temperature. Higher pretreatment conditions lead to
degradation of sugars to hydroxymethyl furfural, which inhibits
yeast growth by reducing the biological enzymatic activities,
causing DNA and cell wall damage, inhibition of RNA and protein
synthesis [53].
Effect of reaction time on hydrolysis
To investigate the release of total sugar and reducing sugar,
E. intestinalis and U. lactuca was hydrolyzed
by 0.7 N and 0.5N H2SO4 respectively at
121oC and different reaction time varying from 15,
30, 60, 90 and 120 min (Fig 4). Maximum total sugar of 399 mg
g-1was obtained at 105 min for E.
intestinalis; maximum reducing sugar (121 mg
g-1) was recorded at 45 min with a conversion
efficiency of 42.1%. Maximum total and reducing sugars were
produced for U. lactuca at 45 min and were seen
decreasing with the increase in incubation time. G.
verrucosa was subjected to pretreatment using 0.1N
H2SO4 and maximum total sugar (12.06g
L-1) and reducing sugar (6.99g L-1) was
obtained at 15 min incubation time. A shorter incubation time is
required for red algae as a major fraction of sugar (i.e.
floridean starch) is composed in the cytoplasm of the red algae
[54], which gets released easily. Hence, the longer incubation
time was not considered as it leads to increase energy and cost,
as well as accelerates the degradation of sugars to 5-HMF,
levulinic acid, and formic acid [10, 13, 32, 45]. Therefore, 45
min was considered as the optimum incubation time for further
studies.
Fig. 2 Reducing sugar release
using diferent concentrations
of acid for U. lactuca and E.
intestinalis (p<0.05)
Fig. 3 Efect of reaction temperature on acid hydrolysis of
U. lactuca and E. intestinalis
(p<0.05)
Fig. 4 Efect of incubation time
on total and reducing sugar
release during acid hydrolysis
from U. lactuca and E. intestinalis (p<0.05)
Effect of substrate concentration on acid
hydrolysis
The effect of varying substrate concentrations (1-9% w/v) on acid
hydrolysis of E. intestinalis and U. lactuca
was investigated at 121oC for 45 min (Fig 5). Total
and reducing sugar concentration decreased with an increase in
substrate concentrations. Similar results were observed for E.intestinalis,
and the total reducing sugar decreased with an increase in solid
to liquid ratio [13]. In a conventional simultaneous
saccharification and fermentation process, substrate
concentration of 10% (w/v) is considered optimal due to high
viscosity and difficulty in handling the slurry [50]. Hydrolysis
of Kappaphycus alvarezii required a 10% substrate
concentration [30]. The highest sugar conversion rate was
achieved at 2% (w/v) of G. verrucosa [29]. Higher
efficiency of 85.43% and 62.97% were obtained for 5% (w/v) of
E. intestinalis and U. lactuca substrate.
Therefore, 5% (w/v) was considered as optimum substrate
concentration.
Optimized sugar from E. intestinalis and U. lactuca
Acid hydrolysis of E. intestinalis and U.
lactuca feedstock was carried out at an optimum acid
concentration of 0.7N and 0.5N H2SO4
respectively at 121oC for 45 min 5% (w/v) substrate
concentration (Table 3). The highest reducing sugar of
206.82±14.96 mg g-1was recorded from
U.fasciata using sodium acetate (pH 4.8) buffer
pretreatment process at 120 oC for 60 min. Undaria
pinnatifida was pretreated at a higher concentration of
acid, 5% H2SO4 at 120 oC for 24
h to obtain 65 mg glucose/g biomass [52]. U. pertusa
was subjected to a high thermal liquefaction process (HTLP),
with a process condition of 400 oC at 40 mPa, and
obtained 352 mg g-1of reducing sugar. HTLP
pretreatment loosens the complex structure and increases the
porosity of the cell membranes allowing the entry of the solvent
for further degradation [55]. Reducing sugar concentration of
145±2.1 mg g-1was obtained from pretreatment of Saccharina
japonica (10% w/v) at 121oC for 60 min using
40 mM H2SO4 [37]. Pretreatment of red
seaweed Gracilaria sp. was carried out using 0.1 N
H2SO4 at 121oC for one h at 20%
w/v biomass loading and obtained 277 mg g-1of
reducing sugar [56]. Inhibitors from acid hydrolysate
hydroxymethyl furfural (HMF) and Levulinic acid (LA) were
detoxified using activated charcoal [45], which removed 70.37%
HMF and 38.8% LA, similarly, Na2CO3
detoxified the 56.1% from U.lactuca and 23.3% from E.intestinalis [44] indicating
that hydrolysis using dilute acid concentration resulted in a
lower concentration of inhibitors.
TLC analysis showed glucose and xylose in the both acid
hydrolysate of E. intestinalis and U. lactuca
(Fig 6).
Assessing the optimal pretreatment conditions
through RSM (Response Surface Method)
RSM involved assessing the optimal pretreatment conditions (Table
4) for maximum reducing sugar yield from E.
intestinalis and U. lactuca. The possible
combinations of independent variables were chosen through
stepwise regression, and the probable relationship with the
yield of sugar (Y) is expressed in equations 2 for E.
intestinalis and 3 for U. lactuca,
respectively (p<0.05). Response surface curves were generated
using 14 data points of each variable as depicted in Fig 7 for
E. intestinalis and U. lactuca reducing sugar
yield at different reaction temperatures, time, and substrate
concentrations, which aided in arriving at the optimum level of
each variable for maximum response. An increase in substrate
concentrations led to a decline in reducing sugar release, which
could be due to sorption loss.
Fig. 5 Efect of substrate concentration of acid hydrolysis of
U. lactuca and E. intestinalis
Table 3 Optimized sugar
release using dilute acid
pretreatment of E. intestinalis
and U. lactuca
Fig. 6 TLC analysis of hydrolysate obtained after optimized
acid hydrolysis of algal biomass; E. intestinalis (EI) and U.
lactuca (UL) with glucose (G)
and xylose (X) as standards
Ye=584.9+4.8X1-4.7X2-5.7X3-0.05X12-0.017X22-1.45X32……
Eq. (2)
Yu=293.2+11.6X1-3.2X2+17.9X3-0.09X12-0.01X22-3.8X32
…...Eq. (3)
The effect of reaction temperature and incubation time on
hydrolysis of E. intestinalis and U.
lactuca when substrate concentration was kept constant
as shown in Fig 7, and reducing sugar yield decreased with an
increase in incubation temperature and incubation time. Higher
reducing sugar yield was recorded at lower temperatures (30-60 º
C) and incubation time (30–90 min). In order to obtain reducing
sugar yield between 400-600 mg/g, the optimum reaction
temperature of 75 ºC, reaction time 75 min, and substrate
concentration of 5% w/v were recorded from the RSM 3D plot.
However, in this study, dilute acid hydrolysis of algal biomass
for efficient reducing sugar yield between 200- 240 mg/g was
achieved at temperature 121 ºC, time 45 min at 5% w/v substrate
concentration. Higher glucose yield for Sargassum spp.is
achieved at optimized acid concentration of 3.75 and 4.5% (w/v)
substrate concentration, and optimum temperature 115 ºC for
86-90 min [35]. It is seen that the pretreatment temperature and
incubation time obtained in this study to treat E.
intestinalis and U. lactuca were milder than
the terrestrial biomass. The presence of cellulose,
hemicellulose, and lignin imparts the rigidity to the
terrestrial biomass. It hence requires a temperature between
165-210 ºC at a high concentration of acids for a longer
incubation time (4 weeks) [35, 37].
It is seen that the pretreatment temperature and incubation time
obtained to treat E. intestinalis and U.
lactuca were milder than the terrestrial biomass
requiring temperature between 165-210oC at a high
concentration of acids for longer incubation time (4 weeks). A
harsh pretreatment condition was required due to the rigidity of
the biomass with cellulose, hemicellulose, and lignin [35, 39].
Estimated effects, standard errors (SE), Student’s t test and
significance value for the model representing reducing sugar
yield from U. lactuca and E. intestinalis
represented in Table 5.
Fig. 7 Response surface plots of reducing sugar yield for E. intestinalis (1a–b) and U. lactuca (2a–b) after dilute acid pretreatment at
diferent reaction temperature, time, and substrate concentrations. 1a
and 2a Reducing sugar yield at substrate concentration=5% w/v; 1b
and 2b reducing sugar yield at temperature=75 °C; 1c and 2c reducing sugar yield at reaction time=75 min (p<0.05)
Enzyme purification and
characterization
The purification of cellulase enzyme is summarized in
Table 6, which is a two-step
purification, includes ammonium sulphate precipitation and size
exclusion chromatography. The purified enzyme exhibited 7.24 U
mg-1 of specific activity, and 61.82% yield was
obtained with 2.97-fold purification. Purification was further
confirmed by observing a single protein band on SDS-PAGE
(Fig 8) with an estimated molecular mass of
29kDa. Similarly, molecular mass was obtained for cellulase
extracted from Salinivirbrio sp. NTU-05 exhibiting 32.4
U mg-1 specific activity and 18.9% recovery with 29.5
fold purification [58]. Extraction of Endo-β-1, 4-glucanase
Cel5A from Vibrio sp. exhibited a molecular mass of
50kDa, indicating functional cellulase gene in Vibrio genus
(Gao et al., 2010).
The enzyme exhibited the highest activity at pH 6, and the
activity profile showed that the enzyme was active over a wide
range of pH, retaining 90% of its activity (Fig
9). Similar characteristic pH tolerance over a wide
range has been studied earlier for Paenibacillus sp.
pH 7 [59]; Marinobacter sp. MS1032 [60];
Vibrio sp. G21 pH 6.5-7.5 [61]; Bacillus sp. H1666 pH
3-9 [62]; Bacillus sp. [63]; Stachybotrys
atra BP-A [64], Bacilus flexus pH 8-12 [65];
Salinivibrio sp. pH 6.5-8.5 [58].
Fig. 8 SDS-PAGE of purifed cellulase from Vibrio parahaemolyticus. Lane 1, protein ladder; lane 2, cellulase enzyme in 10% SDSPAGE
Fig. 10 Efect of diferent temperatures on enzyme activity
The highest activity of the enzyme was recorded at 50 °C with
higher stability between 40-55°C (Fig 10).
Enzyme activity decreased due to the fluidity of protein
conformation with an increase in temperature above 55°C. It was
seen that around 60% of the activity remained at 60°C. The
optimum temperature for cellulase-producing bacteria was 40-60°C
[58–60, 62–65].
Enzyme activity declined with an increase in salt concentration;
at NaCl concentration >10%, the enzyme retained 5% of its
activity after 24 h (Fig 11). Enzyme activity was above 20% up
to 10% NaCl concentration. It is seen that NaCl concentration
induces the activity of endo-β-1, 4-glucanase Cel5A from Vibrio
sp. G21 and EgI-AG from alkaliphilic Bacillus
agaradhaerens [61, 66]. Enzyme displayed activity in a
broad range of 0-10% NaCl concentration with optimum NaCl
concentration of 3%. Enzyme activity was stable only up to 10%
NaCl concentration despite the enzyme being extracted from
marine bacteria V. parahaemolyticus.
Dilute
acid pretreatment and Enzymatic hydrolysis
Acid hydrolysis of E. intestinalis and U.
lactuca biomass was carried out at an optimum acid
concentration of 0.7N and 0.5N H2SO4,
respectively, at a temperature of 121oC for 45min
incubation time and 5% (w/v) substrate concentration
(Table7).
In similar studies, the highest reducing sugar of 206.82±14.96 mg
g-1 was recorded from U.fasciata using
sodium acetate (pH 4.8) buffer pretreatment at 120 oC
for 60 min. Undaria pinnatifida was pretreated at a
higher concentration of acid, 5% H2SO4 at
120 oC for 24 h to obtain 65 mg glucose
g-1 biomass [52]. U. pertusa was subjected
to a high thermal liquefaction process (HTLP), with a process
condition of 400 oC at 40 mPa, and obtained 352 mg
g-1 of reducing sugar. HTLP pretreatment loosens the
complex structure and increases the porosity of the cell
membranes allowing the entry of the solvent for further
degradation [55]. Reducing sugar concentration of 145±2.1 mg
g-1was obtained from pretreatment of Saccharina
japonica (10% w/v) at 121oC for 60 min using
40 mM H2SO4 [37]. Pretreatment of red
seaweed Gracilaria sp. using 0.1N
H2SO4 at 121oC for one h at 20%
w/v biomass loading yielded 277 mg g-1 of reducing
sugar [56].
Enzyme hydrolysis is affected by various factors, such as
temperature, pH, and concentration (enzyme/substrate).
Increasing enzyme concentration will speed up the reaction, as
long as there is substrate availability; however, if once all of
the substrates are bound, the reaction will cease to speed up.
On the other hand, increasing substrate concentration also
increases the rate of reaction to a certain extent. But once all
enzymes are bound, any increase in substrate will have no effect
on the reaction rate due to saturation of available enzymes.
Trivedi et al. (2015) isolated cellulase enzyme from Cladosporium
sphaerospermum and subjected Ulva lactuca, green
seaweed to enzymatic hydrolysis and obtained 112 mg/g of
reducing sugar. However, in this study, the highest reducing
sugar of 107.6 mg/g was obtained from U. lactuca whereas 135.9
mg/g reduced sugar from E. intestinalis indicating
enzyme ability to hydrolyse the macroalgal polysaccharide.
Acid pretreated macroalgal biomass (E. intestinalis and
U. lactuca) were subjected to enzymatic hydrolysis
using purified enzyme and incubated for 24h, and observed
two-fold increase in reducing sugar in both biomass and 1.2-fold
increase from dilute acid pretreatment, compared to crude
enzymatic hydrolysis. Enzymatic hydrolysis of U.lactuca
using purified enzyme extracted from Bacillus sp. H1666
yielded 450 mg g-1 of reducing sugar, indicating the
potential applicability of the enzyme for algal biomass
saccharification [62]. Enzymes secreted from the cell are
generally found along with other proteins, lipids,
polysaccharides, and nucleic acids. The measurement of enzyme
purity is defined as the relation of the activity of the enzyme
to the total protein present (i.e., the specific activity).
Enzyme purification is carried out in order to remove the
contaminants and increase the specific activity [67]. In this
study, purified enzyme yielded higher reducing sugar due to
increased specific activity (Table 6). Scanning
Electron Microscopy
Scanning electron microscopic (SEM) analysis of macroalgal
biomass revealed ultrastructural changes in the biomass during
dilute acid pretreatment. Fig 12 a and 12 b depict the untreated
surface of E. intestinalis and U. lactuca, raw
or untreated biomass had continuous, even, and smooth surfaces.
Whereas biomass after dilute acid pretreatment had loosened the
rugged surface, which increased the surface area, exposing more
internal cellulose for enzymatic hydrolysis. The roughness of
the seaweed surface after dilute acid hydrolysis makes it more
liable for enzymatic hydrolysis. The presence of strong hydrogen
bonding of cellulose and Vander Waal forces of glucose molecules
imparts the crystalline structure to biomass [32, 68]. Scanning
Electron Microscope (SEM) images revealed cracks and holes on
the pretreated algal surface. Gelidium amansii treated
at 121oC were observed under SEM, electron
micrographs revealed fibers exposed in autoclaved samples
allowing enzymes to easily degrade the cells [32].
Fig. 12 a Scanning electron
micrograph of E. intestinalis
depicting ultrastructural changes
in the feedstock — untreated
sample compared with the acid
and enzyme treated. b Ultrastructural changes evident in the
scanning electron micrograph of
U. lactuca macroalgal biomass
— untreated, acid, and enzyme
treated
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