Sargassum angustifolium brown macroalga as a high potential substrate for alginate and ethanol production with minimal nutrient requirement

Algal Research 36 (2018) 29–36

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Sargassum angustifolium brown macroalga as a high potential substrate for alginate and ethanol production with minimal nutrient requirement

T

⁎

Yasaman Ardalana, Mohammadhadi Jazinia, , Keikhosro Karimia,b a b

Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran Industrial Biotechnology Group, Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Sargassum angustifolium Bioethanol Sodium alginate Biorefinery approach

The brown macroalga, Sargassum angustifolium harvested from the Persian Gulf in the summer and winter was exploited within a biorefinery approach for valuable bioproducts and ethanol production. This alga mainly contained alginate (20.9–22.4%), glucan (36.2–43.1%), lignin-like materials (i.e., polyphenols; 11.8–25.8%), and ash (43.8–35.5%). The biomass protein content was highly depended on the harvesting season, where the biomass harvested in summer and winter comprised 11.9% and 19.7% protein, respectively. The alginate was first extracted in the form of sodium alginate. Then, the residual biomass obtained during alginate extraction was used for bioethanol production through the separate hydrolysis and fermentation. The results demonstrated that 44.5 (28.0) to 53.3 (32.5) kg of ethanol and 2.4 × 102 (2.2 × 102) kg of sodium alginate could be obtained from each tone of summer (winter) algal biomass. During the extraction of alginate, a nutrient-rich extract, “macroalgal extract”, was produced. This extract was successful substitute for yeast extract, which was necessary for the ethanolic fermentation. The results revealed the great potential of S. angustifolium for implementation as a source of sodium alginate, a nutrient-rich extract, and bioethanol.

1. Introduction Macroalgae as a source of third generation biofuels has attracted great attention recently. The cell wall of marine algae generally contains three components, i.e., alginic acid, cellulose, and other polysaccharides [1]. The cellulose in the cell wall can be hydrolyzed to sugars and fermented to ethanol. However, the economic feasibility of bioethanol production from macroalgae is still doubtful. Minimization of nutrient cost is one the solutions for improvement of the process economics. Since the cell wall of the macroalgae contains valuable products like alginate, simultaneous production of a biofuel and a value-added product may increase the profitability. Commercial production of alginate, the salt of alginic acid, relies on the brown seaweeds containing alginate in their cell wall. Laminaria sp., Macrocystis sp., and Ascophyllum sp. are the main sources of alginate production in industrial scale. It has versatile applications in pharmaceutical, nutraceutical, and cosmetic industries [2,3]. S. angustifolium [4], S. binderi and Turbinaria ornata [5], S. natans [6], and Padina spp. [7] are among the seaweeds containing appreciable amounts of alginate. The studies on simultaneous production of a biofuel and a valueadded product from macroalgal biomass were rarely reported: Tan and

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Lee [8] studied ethanol production from solid wastes of macroalga Eucheuma cottonii after the extraction of carrageenan. Bioethanol production from red macroalga Gracilaria verrucosa was done by Kumar et al. [9]. They achieved the yield of 0.43 g of ethanol per g of sugar using the residual biomass of algae remained after agar extraction. A biorefinery approach was developed to produce a range of valuable products using the kelp Laminaria digitata. The approach was based on the extraction of alginate and fucoidan and subsequent production of ethanol [10]. It has been reported that alginate industries produce leftover pulps that can be the used as a feedstock for bioethanol production [11]. In order to produce bioethanol from macroalgae, the biomass must be pretreated before. Different types of pretreatment strategies have been implemented so far: Yazdani et al. [12] produced bioethanol from brown macroalga Nizimuddinia zanardini. They obtained 30.5 and 34.6 g of ethanol per kg of dried algae in acidic and hot water pretreatment, respectively. Brown macroalga Saccharina japonica was used as a substrate for the production of ethanol after acidic pretreatment by Lee et al. [13]. They converted cellulosic materials into glucan and observed 67.4% as the yield of bioethanol from glucan. Kim et al. [14] investigated bioethanol production from macroalga

Corresponding author. E-mail address: [email protected] (M. Jazini).

https://doi.org/10.1016/j.algal.2018.10.010 Received 20 February 2018; Received in revised form 10 October 2018; Accepted 10 October 2018 2211-9264/ © 2018 Elsevier B.V. All rights reserved.

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washed with distilled water to remove remaining HCl. The filtrate was designated as algae extract 1 (ALEX 1) and used as a nitrogen source in the subsequent fermentation. Afterward, 250 ml of sodium bicarbonate solution (2% w/w) was added to the filter cake and put into a water bath at 90 °C for 2 h. Subsequently, the mixture was filtered and the filter cake (algal biomass) was dried in an oven at 45 °C for 24 h and weighed. This was the weight of residual macroalga after alginate extraction. This residual biomass was used for bioethanol production. The filtrate was added to 375 ml 0.2 M HCl to form the insoluble alginic acid. The suspension was then filtered. The filtrate was designated as algae extract 2 (ALEX 2) and used as a nitrogen source for the subsequent fermentation. The filter cake (alginic acid) was mixed with 250 ml of sodium carbonate solution (2% w/w) to convert insoluble alginic acid to soluble sodium alginate. Finally, 250 ml of ethanol was mixed with the mixture to form insoluble alginate. Then, the solids were filtered and subsequently subjected to hot air at 45 °C for 24 h. The weight of sodium alginate was measured and used for the calculation of sodium alginate yield according to the following equation [17]:

Laminaria japonica. They applied acidic and subsequently enzymatic pretreatment. They cultivated recombinant Escherichia coli KO11 on the hydrolysate. They reported the yield of 0.4 g ethanol per g of sugars. Obtaining the high yield was related to the ability of bacteria for the utilization of mannitol in the hydrolysate. Borines et al. [15] studied bioethanol production from Sargassum spp. They used acidic and subsequently enzymatic pretreatment. Then the hydrolysate was subjected to fermentation. They found the yield of ethanol from sugars as 89% which was much higher than the theoretical yield of bioethanol from glucose (51%). They pointed out that this was due to the conversion of sugars other than glucose to ethanol. The pretreatment of brown algae Macrocystis pyrifera biomass followed by enzymatic saccharification has been reported recently [16]. The pretreatment was performed using dilute sulfuric acid and ionic liquids. In the extraction of alginate from macroalgae, acid was applied to the biomass (Section 2.2). This may act as a kind of acid pretreatment similar to acid pretreatment methods presented above. Therefore, extraction of a valuable product from macroalgal biomass, may not only increase the profitability but also disrupt the recalcitrant structure of the cell wall. In addition, the liquor obtained during extraction contains nitrogen and could be utilized in the subsequent fermentation step. Hence, the idea behind this work was to produce a valuable product (sodium alginate) in addition to bioethanol, as a biofuel, from macroalgal biomass. In order to reduce the cost of nutrients, it was aimed at exploitation of the liquor obtained after extraction of alginate for the fermentation. To the knowledge of the authors, this is for the first time that the production of bioethanol and alginate from S. angustifolium, an abundant macroalgae in Persian Gulf was studied. Additionally, the strategy used in this work for minimization of nutrient requirements is novel. This work paved the way for the production of third generation ethanol through a cost-effective process. Doing so, alginate and ethanol were produced under a biorefinery framework as presented in Fig. 1.

Sodium alginate yield (%) =

weight of dried sodium alginate × 100 weight of dried macroalgae powder (1)

The recovery of solid residue was defined as follows:

Recovery of solid residue (%) =

weight of dried solid residue × 100 weight of dried macroalgae powder (2)

The quality of produced sodium alginate was analyzed using Fourier transform infrared spectroscopy (FTIR, Bruker Tensor 27 FT-IR, Billerica, MA, USA). A commercial sodium alginate (Sigma-Aldrich, CAS Number: 9005-38-3) was used for comparison purposes.

2. Materials and methods 2.1. Biomass collection and preparation

2.3. Compositional analyses of biomass

S. angustifolium, a Persian Gulf native macroalga, was purchased from Algae Biotechnology Center of the Persian Gulf, Bushehr, Iran (Species ID: ABCM007). The alga was harvested from Naftkesh zone, Bushehr, Iran (longitude: 50.808244, latitude: 28.999055, and height from sea level: −1 m). In order to compare the effects of atmospheric conditions at which alga grew on the production of ethanol and alginate, the macroalga was harvested in both summer and winter. The harvested macroalga was washed with tap water to remove salts and sands, and then dried at ambient temperature. The dried alga was pulverized and sieved using 80 mesh screen to achieve particle size of less than 0.177 mm.

Standard method prepared by the National Renewable Energy Laboratory (NREL, Denver, United States) was used for determination of carbohydrate and lignin content of raw algae as well as the residual solid remained after alginate recovery [18]. Protein content was also measured according to the method developed by Johann Kjeldahl [19]. Ash content was measured following the procedure provided by NREL [20]. Accordingly, a small amount of sample was put into a previously weighed crucible and weighed. It was heated at 105 °C in an oven for 4 h and then the sample was weighed. Total solid content of the samples was calculated based on the difference in the weight of the sample after and before heating. Next, the sample was ignited in a furnace at 550 °C for 1 h. After cooling in a desiccator, the weight of the sample was measured. The Ash content was calculated according to the difference in the weight of sample after and before ignition. The amount of nitrogen element exist in the biomass was measured using CHNSO Analyzer (Elementar, Vario EL III, Germany).

2.2. Sodium alginate extraction and qualification An amount of 10 g of dried macroalga was added to 75 ml of 0.2 M HCl and agitated. The mixture was then filtered and the solids were

Fig. 1. Schematic view of the biorefinery approach implemented in the present study for full exploitation of macroalgae S. angustifolium biomass. 30

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Table 1 (a) The yield of sodium alginate and recovery of solid residue obtained from summer and winter macroalgal biomass and (b) total solid content measured for raw and residual biomass as well as algal extracts 1 and 2. Data shown is the mean ± SD, n = 3.

produced ethanol Ethanol yield (%) =

Yield of sodium alginate (%)

Recovery of solid residue (%)

Summer Winter

24.4 ± 1.0 22.4 ± 0.8

31.0 ± 0.9 16.5 ± 0.7

g l

1.111 × 0.51 × biomass concentration

( )×f g l

× 100 (4)

The coefficient 0.51 is the theoretical yield of ethanol from glucose. Each mole of glucose (180 g) can be converted to 2 mol of ethanol (2 × 46 g); thus, the theoretical yield is 2 × 46/180 = 0.51 g/g. All experiments and measurements were performed at least in triplicate. The standard deviation was shown as the error bar.

a Harvesting time

()

2.5. Analytics b Harvesting time

Summer Winter

A high-performance chromatography system (HPLC) equipped with UV/VIS and RI detectors (Jasco International Co., Japan) were used for ethanol measurement. An Aminex HPX-87H column (Bio-Rad, USA) at 60 °C with 0.6 ml/min eluent of 5 mM sulfuric acid was implemented. Total reducing sugars were determined according to the protocol proposed by Miller [25].

Total solid content (%) Raw biomass

Residual biomass

ALEX 1

ALEX 2

89.6 ± 0.1 92.5 ± 0.1

96.2 ± 0.6 96.7 ± 0.6

3.66 ± 0.01 5.90 ± 0.03

0.821 ± 0.002 0.832 ± 0.002

2.6. Statistical method 2.4. Ethanol production

One-way analysis of variance (ANOVA) was implemented to investigate the significance of the observed differences at 5% significance level.

Both residual and raw macroalgal biomass were subjected to separate hydrolysis and fermentation for ethanol production. Since there is considerable amount of glucan in the algal biomass, the state of art commercial hydrolytic enzymes, namely cellulase and hemicellulose, Cellic® HTec2 and Cellic® CTec2 (Novozymes, Denmark), used for the production of simple sugars from the biomass. Cellic CTec2 and Cellic HTec2 in a volume ratio of 9:1 were mixed [22]. According to the method provided by NREL [23], the activity of enzymes was 125 FPU/ ml for Cellic CTec2 and 23 FPU/ml for HTec2. The experiments were done in 118 ml glass bottle. A 40 ml medium containing of 25 g/l of either raw or residual macroalgae biomass supplemented with: NH4SO4, 7.5 g/l; K2HPO4, 3.5 g/l; CaCl2·2H2O, 1 g/l; MnSO4, 0.75 g/l; and yeast extract or algae extract, 5 g/l; was prepared in 0.5 M sodium citrate buffer. The pH of the solution was adjusted to 4.8 using 1 M HCl, and then the bottle was autoclaved for 20 min at 121 °C. After cooling to room temperature, 20 FPU of the enzyme mixture per gram of algal biomass was added to the mixture under aseptic conditions. Afterward, the bottle was put into a shaking incubator for 72 h. The temperature and rotation speed were set to 45 °C and 120 rpm, respectively. Samples were periodically taken to analyze the glucose concentration. Then, the yield of enzymatic hydrolysis was calculated [24]:

3. Results and discussion In this section, the extraction and qualification of alginate from the alga was first presented. Then the characterization of macroalgal biomass was presented and discussed. Afterward, the results of fermentation and ethanol production were described. At the end, the overall mass balanced on the proposed process was demonstrated. 3.1. Extraction and qualification of alginate Table 1-a shows the yield of sodium alginate obtained from summer and winter macroalgal biomass. It was observed that the yield was 24.4 and 22.4% for summer and winter, respectively. This means that the potential of macroalgae harvested in summer for alginate production was slightly higher than that harvested in winter. The yield of residual biomass was also presented in this table. The recovery of residual biomass from the summer sample was about two-fold greater than that obtained from the winter sample (31% compared to 16.5% obtained from summer and winter biomass, respectively). The recovery of the process highly depends on the composition of substrate. As mentioned in Section 2.2, the extraction process involved an acid treatment. Typically, glucan is the most resistant part of biomass to acid treatment, while mannan, galactan, and xylan can be easily hydrolyzed to liquid phase [26]. Thus, the higher recovery of residual biomass from the summer sample was related to its higher glucan content. Glucan content of raw summer and winter macroalgal biomass were measured as 43.1 and 36.2%, respectively (Table 2). Basha et al. [17] reported 17.5 and 21% sodium alginate yield from Sargassum subrepandum extracted at 25 and 80 °C, respectively. The production of sodium alginate from Sargassum fluitans and Sargassum oligocystum were investigated by Davis et al. [27]. They used sodium carbonate for the alginate extraction from S. fluitans and obtained the sodium alginate yield of 22.8 and 24.4% residual biomass recovery. The corresponding values for S. oligocystum were 18.5 and 17.8%. Mazumder et al. [28] investigated the extraction of sodium alginate from Sargassum muticum. They observed 13.6% yield of alginate. The alginate yield obtained in this study demonstrated the high potential of S. angustifolium for the production of sodium alginate. Total solid content of residual biomass obtained from summer and winter samples was almost the same (Table 1-b: 96.2 compared to

Yield of enzymatic hydrolysis (%) produced glucose =

() g l

1.111 × biomass concentration

( )×f g l

× 100 (3)

where f was the percentage of glucan in the biomass. The factor of 1.111 is the hydration factor of glucan. In fact, when glucan, (C6H10O5)n (with molecular weight of n × 162) is hydrolyzed to (n) glucose (C6H12O6) with molecular weight of 180 (n ∗ 180), the total weight will increase by 180/162 = 1.111. This is referred to “hydration factor of glucan”. The fermentations were conducted with and without addition of yeast extract as well as with ALEX 1 and ALEX 2, as the substitute for yeast extract. After enzymatic hydrolysis (24 h), the mixture was inoculated with 1 g/l of S. cerevisiae. Then, the bottle was sealed with a butyl rubber and an aluminum cap. Nitrogen was purged to attain anaerobic conditions in the bottle. Next, it was put into an incubator at 37 °C for 48 h. Samples were periodically taken and the concentration of produced ethanol was measured. The yield of ethanol was calculated according to the following equation [24]: 31

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96.7%). This was also observed in the case of ALEX 2 (0.822 compared to 0.831% for summer and winter samples, respectively). The lower total solid content in the raw summer macroalgae compared to the winter one, was in accordance with total solid content in ALEX 1, which was much lower for summer macroalgal biomass compared to winter macroalgal biomass (3.6 compared to 5.9%). Fig. 2 shows the FTIR spectrum of the control (commercial sodium alginate) as well as the sodium alginate extracted from summer and winter samples. The peaks at 3421, 2942, 1611, and 1416 cm−1 wavenumbers were assigned to hydrogen bonded OeH stretching vibrations, CeH stretching vibrations, asymmetric stretching of carboxylate OeCeO vibration, and symmetric stretching vibration of the carboxylate group, respectively [29,30]. The spectrums showed that the peak values at these wavenumbers for winter and summer macroalgal biomass are close to those of commercial sodium alginate. At 3421, 2942, 1611, and 1416 cm−1, the values were (0.87, 0.94, 0.83), (0.25, 0.38, 0.3), (0.91, 1.04, 0.89), and (0.65, 0.89, 0.74) for control, summer, and winter samples, respectively. The close proximity of the peak values of produced sodium alginate to those of commercial one reveals that the chemical structure of the product was similar to the commercial one. In addition, the peak values showed that the alginate prepared from winter sample had more similar quality to the commercial sample since chromatogram was much closer to the commercial one.

Table 2 Composition of raw and residual macroalgal biomass (%) harvested in winter and summer. Data shown is the mean ± SD, n = 3. Material

Glucan Xylan Galactan Mannan Total lignin Alginic acid Protein Ash

Summer macroalgal biomass

Winter macroalgal biomass

Raw biomass

Residual biomass

Raw biomass

Residual biomass

43.1 ± 1.2 0 2.5 ± 0.8 1.7 ± 0.3 25.8 ± 0.3 22.4 ± 0.9 11.9 ± 0.7 35.5 ± 0.6

50.0 ± 1.4 0 3.0 ± 0.1 1.9 ± 0.1 13.3 ± 0.4 – 13.7 ± 0.8 –

36.2 ± 0.1 0 2.1 ± 0.1 1.2 ± 0.1 11.8 ± 0.6 20.9 ± 0.8 19.8 ± 1.1 43.8 ± 0.5

49.5 ± 1.3 0 2.9 ± 0.1 1.9 ± 0.1 6.0 ± 0.6 – 20.1 ± 1.2 –

a

3.2. Biomass composition and characterization The composition of macroalgal biomass before and after extraction of sodium alginate is summarized in Table 2. As shown in this table, all samples are free of xylan. This is also confirmed by results obtained via HPLC (data not shown). Moreover, there was no xylose in the hydrolysate obtained after acidic hydrolysis. However, Yazdani et al. [12] reported that Nizimuddinia zanardini, another microalga widely available in the same area that the current study microalga was obtained, contains 30 g xylan per kg of dried biomass. The glucan content observed in S. angustifolium (43.1 and 36.2% for summer and winter samples respectively) was much higher than that observed in N. zanardini (10%) [12]. Despite the high glucan content of the macroalgal biomass, galactan and mannan contents were negligible. They were available in the order of only 2–3%. Small amount of mannose and galactose in the hydrolysate of acidic treatment (data not shown) confirmed the existence of tiny amount of mannan and galactan in the biomass. Ge et al. [31] found similar results in the pretreatment of Laminaria japonica macroalgae. They showed that there was no galactose, xylose, and mannose in the hydrolysate. The sum of ash, protein and total carbohydrate content of raw biomass was more than 100% (Table 2). This is because of the fact that the implemented measurement method (Section 2.3) has some inaccuracy when the substrate contains considerable amounts of proteins and extractives. This method cannot distinguish between “lignin” and “lignin-like materials”. Similar problem was reported and thoroughly discussed by Taherzadeh et al. [32]. High amount of lignin (25.8%) was measured in the summer macroalgal biomass which was reduced considerably after the extraction of sodium alginate (25.8 to 13.3%). This revealed that washing of macroalgae with acids removed lignin from the biomass. However, it should be noticed that the cell of S. angustifolium does not contain any lignin [15]. Nevertheless, they contain lignin-like materials, which can be removed during washing with acids. Therefore, lower lignin content is observed after extraction of sodium alginate. Yazdani et al. [12] reported that N. zanardini contains 12.3% lignin. This is similar to the winter sample which contains 11.8% lignin. The summer and winter macroalgal biomass contained 35.5 and 43.8% ash, respectively (Table 2). The measured ash content was higher than the values reported for other macroalgae. For instance, Ulva lactuca, Gelidium amansii, and Sargassum fulvellum contain 18.9, 8.6, and 31.5 ash, respectively [14]. The ash content of S. horneri was 12.8 to 36.7% [33] depending the harvesting season. Difference in algal ash

b

c

Fig. 2. FTIR spectrum of (a) commercial sodium alginate, (b) sodium alginate obtained from summer macroalgal biomass, and (c) sodium alginate obtained from winter macroalgal biomass.

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ANOVA, the prolongation of enzymatic hydrolysis resulted in no significant improvement of the yield for all samples, except for the residuals from the summer samples. Comparison of the yields of enzymatic hydrolysis obtained after 24 h with those obtained after 72 h resulted in F-value of 17.4004 (p-value = 0.0529) and 13.5494 (pvalue = 0.0665) for raw summer and winter samples, respectively, whereas the critical F-value was 18.5128. However, the F-value and pvalue for residual biomass of summer sample were 1.0231 and 0.4182, respectively. Jelynne et al. [35] reported that the yield of hydrolysis of Sargassum sp. can be enhanced about 23% by pretreatment with 1% sulfuric acid. Acidic pretreatment of brown macroalgae Macrocystis pyrifera with 2% sulfuric acid improved the yield of enzymatic hydrolysis [16]. The increase in ethanol yield after the extraction of sodium alginate means that extraction acted as a kind of pretreatment. The treatment of macroalga with the alkaline solution could destruct the recalcitrant structure of the biomass, providing more accessible sites to the hydrolytic enzymes for glucan hydrolysis. Glucan is usually surrounded by hemicellulosic polymers and lignin-like materials. They act as barriers between cellulase and glucan, particularly more lignin-like materials content results in more barriers to access glucan. The highest yield of enzymatic hydrolysis was observed in the sample with the least lignin content (Table 2, residual winter macroalgal biomass). Hence, the extraction of sodium alginate was not only given the valuable product of sodium alginate but also acted as a kind of pretreatment without any need to perform a separate pretreatment step. After hydrolysis, the hydrolysate obtained from winter and summer macroalgal biomass (raw and residual biomass) was subjected to fermentation. The concentration of ethanol obtained in each fermentation is depicted in Fig. 4. The yield of ethanol was calculated accordingly and presented in Table 3. As illustrated in Fig. 4, the concentration of ethanol reached to the maximum after about 30 h in all experiments. After 30 h of fermentation, there was no significant change in the concentration of ethanol. Therefore, the concentration at 72 h was used for the calculation of ethanol yield. The maximum concentration of ethanol achieved after 72 h fermentation of raw winter and summer macroalgal biomass were in the range of 2.2–3.1 and 3.6–4.0 g/l, respectively (Fig. 4a and b). However, those values for residual winter and summer biomass were in the range of 2.8–3.4 and 4.2–4.9 g/l, respectively (Fig. 4c and d), meaning that the higher concentration of ethanol can be achieved when residual biomass was used. These results were in accordance with the yields calculated in Table 3. The lowest yield and concentration of ethanol were observed in the experiments where no yeast extract was used or ALEX 1 was applied (Table 3). For example, the yield of ethanol from summer residual biomass was 50.1 and 50.7% in the experiments without yeast extract and in the experiment with ALEX 1, respectively. This means that ALEX 1 does not have effective nutritional values. Although the ethanol concentration and yield were lower in the case of ALEX 2 compared to the fermentation at which yeast extract was used, they were higher than the fermentations with ALEX 1 (Table 3). This indicated that ALEX 2 contained more nutritional value than ALEX 1. Therefore, ALEX 2 was preferred to ALEX 1 as the substitute for yeast extract. The results presented in Table 3 shows that the yield of ethanol was higher when residual biomass was used for fermentation. This was due to the fact that the yields of enzymatic hydrolysis in those experiments were higher (Fig. 3), resulting in higher ethanol yields. According to the results presented in Table 3, the yield of ethanol per dried biomass was 0.124 and 0.172 g/g for summer macroalgal biomass and their residual, respectively. However, this yield was 0.136 and 0.187 g/g for the winter sample and their residual, respectively. These yields have the same order of magnitude as the yields reported in the literature. For example, Kumar et al. [9] found out that 0.148 g ethanol can be obtained from 1 g of dried macroalgae Gracilaria verrucosa after extraction of agar. The yield of ethanol per dried biomass of Sargassum sp. after

content is due to the mineral salts attached to the surface of macroalgae. This is in turn related to the environmental conditions at which macroalga was cultivated. For example, macroalgae grown in marine water have more ash content compared to those live in freshwater [34]. As is shown in Table 2, the protein content of the summer macroalgal biomass and its residual were 11.9 and 13.7%, respectively. The protein content of winter macroalga and its residual biomass were 19.8 and 20.1%, respectively. These results are in accordance with the elemental analysis performed for raw and residual macroalgal biomass: The nitrogen content in the summer sample was lower than its residual (1.26 compared to 1.62%). This was similar to that observed for winter macroalgae (2.5 versus 2.8%). Hence, the elemental analysis confirmed the measured protein contents. It was reported that Gracilaria cervicornis and Sargassum vulgare macroalgae contain 19.7 and 13.6% protein, respectively [33]. The nitrogen content of the macroalgae as well as their protein content obtained at different seasonal conditions are in good agreement with carbohydrate content. As presented in Table 2, the sum of glucan, lignin, mannan, and galactan content of the summer sample (73.1%) is more than that for residual biomass (68.2%). However, the protein content of the summer sample was 11.9%, while that of the residual biomass was 13.7%. Similar results obtained for winter macroalgae (Table 2). It was reported that carbohydrate and protein content of macroalgae are inversely related. This might be related to the growing conditions, e.g., light intensity, temperature, and nitrogen content, that inversely affect the protein content which were directly related to carbohydrate content [33]. In other words, winter conditions (low light intensity, low temperature) might be the reason for higher protein content and lower carbohydrate content in the winter sample compared to the summer sample. As presented in Table 2, alginic acid obtained from the summer macroalgal biomass (22.4%) was higher than that obtained from the winter sample (20.9%). This might be due to the fact that the sum of carbohydrates in the summer macroalgae (73.1%) was higher than the sum obtained in winter macroalgae (51.4%), indicating that the summer macroalgae has higher potential to be used as a source of alginate production.

3.3. Enzymatic hydrolysis and fermentation

Yield of enzymatic hydrolysis (%)

Fig. 3 illustrates the yield of enzymatic hydrolysis obtained after and before extraction of alginate. As it is shown in this figure, the yield of hydrolysis increased from 32.1 to 53.2% when the summer macroalgal biomass was subjected to alginate extraction. A similar trend was observed for winter macroalgal biomass (Fig. 3). According to one-way 70

Aer 24 h Aer 72 h

60 50 40 30 20 10 0

Residual biomass (winter sample)

Residual biomass (summer sample)

Raw biomass Raw biomass (summer (winter sample) sample)

Fig. 3. Yields of enzymatic hydrolysis of raw and residual macroalgal biomass obtained from summer and winter samples. Data shown is the mean ± SD, n = 3. 33

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4

4 Ethanol concentration (g/l)

Ethanol concentration (g/l)

a

3.5 3 2.5 2 1.5 1

Raw summer biomass- no yeast and algal extract Raw summer biomass- with yeast extract Raw summer biomass- with ALEX 1 Raw summer biomass- with ALEX 2

0.5

3 2.5 2 1.5

1

20

40 time (h)

60

0 0

80

5

20

40 time (h)

60

80

6 Ethanol concentration (g/l)

c

4.5 Ethanol concentration (g/l)

Raw winter biomass- no yeast and algal extract Raw winter biomass- with yeast extract Raw winter biomass- with ALEX 1 Raw winter biomass- with ALEX 2

0.5

0 0

b

3.5

4 3.5 3 2.5 2 1.5 Residual summer biomass- no yeast and algal extract Residual summer biomass- with yeast extract Residual summer biomass- with ALEX 1 Residual summer biomass- with ALEX 2

1 0.5

d

5 4 3 2

Residual winter biomass- no yeast and algal extract Residual winter biomass- with yeast extract Residual winter biomass- with ALEX 1 Residual winter biomass- with ALEX 2

1 0

0 0

20

40 time (h)

60

80

0

20

40 time (h)

60

80

Fig. 4. The concentration of ethanol during 72 h fermentation of raw and residual biomass obtained from winter and summer macroalgal biomass; (a) raw summer biomass, (b) raw winter biomass, (c) residual summer biomass, (d) residual winter biomass. Data shown is the mean ± SD, n = 3.

obtained in winter macroalgae, the ALEX 2 derived after extraction of sodium alginate showed more nutritional value than ALEX 2 obtained from summer macroalgae. This can be clearly observed in the ethanol yield using ALEX 2 extracted from winter macroalgae (Table 3: 70.3%). As it is shown in Fig. 5, substitution of yeast extract with ALEX 2 resulted in higher ethanol production in the case of winter macroalgal biomass (32.5 kg compared to 30.8 kg). However, use of ALEX 2 instead of yeast extract led to lower ethanol production (49.5 kg compared to 53.3 kg).

Table 3 The yield of ethanol obtained through the separate hydrolysis and fermentation of raw and residual macroalgal biomass. Data shown is the mean ± SD, n = 3. Raw biomass

Residual biomass

Summer macroalgal biomass Nitrogen source No nitrogen source (no yeast extract) With yeast extract Algal extract 1 (ALEX 1) Algal extract 2 (ALEX 2)

Ethanol yield (%) 36.1 ± 1.8 51.1 ± 3.8 37.5 ± 1.3 41. 9 ± 0.9

Ethanol 50.1 ± 60.7 ± 50.7 ± 56.3 ±

yield (%) 0.5 2.2 1.0 1.4

Winter macroalgal biomass No nitrogen source (no yeast extract) With yeast extract Algal extract 1 (ALEX 1) Algal extract 2 (ALEX 2)

54.3 66.8 54.7 63.0

60.5 66.9 60.7 70.3

2.0 1.1 0.7 3.9

± ± ± ±

1.7 3.5 0.8 3.6

± ± ± ±

4. Conclusions S. angustifolium is a suitable source for alginate, fermentable sugar, and ethanol production. Winter macroalgal biomass showed more potential for the production of bioethanol (about 73%). Nevertheless, the yield of sodium alginate obtained from the summer macroalgal biomass was higher (about 9%). Bioethanol production from residual biomass was higher than raw biomass due to the fact that the extraction of alginate acted as a kind of pretreatment. Therefore, there was no need for the separate pretreatment of biomass. One of the expensive nutrients required for ethanol production, i.e., yeast extract, can be substituted with macroalgal extract, resulting in similar bioethanol yield.

acidic pretreatment with 1% sulfuric acid was reported as 0.112 through SSF process [35]. Pretreatment of L. japonica with 0.1% sulfuric acid resulted in 0.143 g ethanol per g of dried biomass [31]. According to the results obtained in this study, the amount of product (both sodium alginate and ethanol) which can be attained from winter and summer macroalgal biomass were calculated based on one tone of dry macroalgae and presented in Fig. 5. Although the laboratory data cannot be directly translated into large scale, the calculated overall mass balance is just to demonstrate the potential of the macroalgae for production of ethanol and sodium alginate. Accordingly, the amount of products produced out of the summer sample was higher than that of winter one (244 kg sodium alginate and 53.3 g ethanol versus 224 kg sodium alginate and 30.8 kg ethanol out of summer and winter samples, respectively). The high amount of ethanol obtained per ton of summer macroalgal biomass is because of the higher amount of residual biomass attained after alginate extraction (Fig. 5: 310 kg and 165 kg in summer and winter samples respectively). Although lower residual biomass

Acknowledgments The financial support provided for this project by Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, is gratefully acknowledged. The authors also grateful to Novozymes, Denmark, for providing Cellic® HTec2 and Cellic® CTec2 enzymes for this study.

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a

b

Fig. 5. Overall mass balance for the production of sodium alginate and ethanol from brown macroalga S. angustifolium

Declaration of author's contributions The experiments were conducted by Mrs. Yasaman Ardalan and supervised by Dr. Keikhosro Karimi and Dr. Mohammadhadi Jazini. The manuscript prepared by Dr. Mohammadhadi Jazini and revised by Dr. Keikhosro Karimi.

[6]

[7]

Conflicts of interest

[8]

The authors declare that they have no conflicts of interest to this study.

[9]

[10]

Statement of informed consent, human/animal rights

[11]

No conflicts, informed consent, human or animal rights are applicable for this study. All authors confirmed the manuscript authorship and agreed to submit it for peer review.

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