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.

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 121^oC 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.

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 (165-210^oC), 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 121^oC 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].

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 121^oC 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)

The effect of varying substrate concentrations (1-9% w/v) on acid hydrolysis of E. intestinalis and U. lactuca was investigated at 121^oC 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.

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 121^oC 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 121^oC for 60 min using 40 mM H₂SO₄ [37]. Pretreatment of red seaweed Gracilaria sp. was carried out using 0.1 N H₂SO₄ at 121^oC 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).

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 165-210^oC 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)

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.

Acid hydrolysis of E. intestinalis and U. lactuca biomass was carried out at an optimum acid concentration of 0.7N and 0.5N H₂SO_4, respectively, at a temperature of 121^oC 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 121^oC for 60 min using 40 mM H₂SO₄ [37]. Pretreatment of red seaweed Gracilaria sp. using 0.1N H₂SO₄ at 121^oC 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 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 121^oC 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