Extraction of inorganic materials from fresh and dried alga Saccharina japonica

Accepted Manuscript Title: Extraction of inorganic materials from fresh and dried alga Saccharina japonica Authors: Patrick Boakye, Divine D. Sewu, Hee Chul Woo, Jae Hyung Choi, Chul Woo Lee, Seung Han Woo PII: DOI: Reference:

S2213-3437(17)30414-1 http://dx.doi.org/10.1016/j.jece.2017.08.030 JECE 1825

To appear in: Received date: Revised date: Accepted date:

24-4-2017 16-8-2017 20-8-2017

Please cite this article as: Patrick Boakye, Divine D.Sewu, Hee Chul Woo, Jae Hyung Choi, Chul Woo Lee, Seung Han Woo, Extraction of inorganic materials from fresh and dried alga Saccharina japonica, Journal of Environmental Chemical Engineeringhttp://dx.doi.org/10.1016/j.jece.2017.08.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Extraction of inorganic materials from fresh and dried alga Saccharina japonica

Patrick Boakye a, Divine D. Sewu a, Hee Chul Woo b, Jae Hyung Choi b, Chul Woo Lee a, Seung Han Woo a, *

a

Department of Chemical and Biological Engineering, Hanbat National University, 125

Dongseo-daero, Yuseong-gu, Daejeon 34158, Republic of Korea b

Department of Chemical Engineering, Pukyoung National University, 365 Sinseon-ro,

Nam-gu, Busan 48513, Republic of Korea

* Corresponding author. Tel: 82-42-821-1537; fax: 82-42-821-1593. E-mail address: [email protected] (S. H. Woo)

Abstract Extraction of minerals from fresh and dried macroalgae kelp (Saccharina japonica) was investigated to get better biomass resource for biorefinery. At a solid to liquid ratio of 1:6 (w/v), 2 h extraction, and 30 oC, inorganic extraction efficiency (Einorg) and total efficiency (Etot) using water were respectively 76.88 and 50.82% for fresh biomass while those of dried biomass were 72.99 and 65.79%. For fresh biomass extraction using ethanol, Einorg (74.19%) and Etot (42.21%) were much higher than for dried biomass with 7.29% Einorg and 1.21% Etot. With 10% ethanol, Einorg were similar for both materials, however, higher ratio of inorganic

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to organic extraction efficiency (rE) (5.48) were obtained for fresh biomass compared to lower rE (2.02) for dried biomass. The rE for fresh biomass was higher (13.80) than that for dried biomass (1.32) using water at 1:4 solid to liquid ratio, suggesting that fresh kelp is better feedstock for bioenergy production.

Abbreviations

DWFS: dry weight of feedstock before extraction DWE: dry weight of extract after extraction

Etot: extraction efficiency for total mass of extract from feedstock Einorg: extraction efficiency for inorganic materials represented by the mass of ash in the feedstock

Eorg: extraction efficiency for organic materials represented by the mass of organics in the feedstock finorg,FS: fraction of ash in feedstock forg,FS: fraction of organics in feedstock finorg,E: fraction of inorganics in extract after extraction forg,E: fraction of organics in extract after extraction finorg,R: fraction of inorganics in residue after extraction forg,R: fraction of organics in residue after extraction fE: mass fraction of extract after extraction

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fR: mass fraction of residue after extraction rE: ratio of Einorg to Eorg

Keywords: Biomass; Biorefinery; Extraction; Inorganics; Macroalgae; Minerals

1. Introduction

Due to the scarcity of resources and energy depletion, there is the need to look out for possible alternative feedstocks for bioproducts and energy production [1]; [2]; [3]. Macroalgae have recently attracted attention as a possible feedstock for biorefinery due to their relatively fast growth rate with high photosynthetic abilities (6-8%) compared to terrestrial biomass (1.8-2.2%) [4]. Considerably, sea-farming scale for mass cultivation of macroalgae can be higher than terrestrial biomasses [5]. Moreover, there is no competition for land and fresh water usage [6]. Compared to terrestrial lignocellulosic biomass, macroalgae contain distinctive compositional properties such as some specific carbohydrates including carrageenan, alginate, agar, and laminarin [5]. Unlike other energy crops such as corn and wheat, macroalgae have a lower risk for food versus energy since their markets are mainly in a few East Asia countries where they are used for food, animal feed and hydrocolloids [7]; [8]. Extracts of macroalgae have also been a source of nutrient-rich fertilizers for crop production [9]; [10]. Environmentally, macroalgae have superior CO2 fixation capabilities than land biomass [11]; [12]; [13]; [14]. Since macroalgae uptake and accumulate several elements found in seawater over their

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whole body, they tend to have relatively higher mineral contents as compared to land based biomass or feedstocks [15]; [16]. Most of these mineral elements are deemed industrially precious and are needed to sustain the economy, especially in the area of electronics. Some of these elements including nickel (Ni), magnesium (Mg), strontium (Sr), lithium (Li), bismuth (Bi), indium (In), molybdenum (Mo), etc., are very abundant in some macroalgae but are rare from land reserves and could face inadequate production risk [15]. Moreover, oceans are the ultimate repository of many materials eroded or dissolved from the land surface. Hence, oceans contain vast quantities of those minerals. Macroalgae are so-called natural adsorbents for these minerals from oceans and consequently have much higher mineral concentrations than oceans do. For instance, Laminaria japonica (brown seaweed) has total mineral contents by up to 451 times higher than seawater [17]. Many products with functional properties including polysaccharides, proteins, poly unsaturated fatty acids, pigments, polyphenols, minerals, plant growth hormones, and others have been extracted from macroalgae over the past decades [18]; [19]; [20]; [21]. For instance, the distinctive specific polysaccharides have been identified as the main substrate for bioenergy production in the form of biofuels through thermochemical means (pyrolysis), chemical treatments, microbial pathways or a combination of both chemical and microbial treatments. Some studies have shown that the presence of higher inorganic metal content of macroalgae has been found to produce a significant catalytic effect on pyrolysis which in turn affects the product yield and bio-oil properties [16]; [22] as well as the convenient continuous operation of reactors [23]. Moreover, the high ash content of macroalgae and the presence of

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metal ions have been found to result in an overall yield of bio-oil derived from the pyrolysis of macroalgae to be lower than that from lignocellulosic biomass [24]; [22]. Typically, high content of potassium was found to cause high char yields and rather reduced bio-oil yields [22]; [25]. Again, one of the major problems often associated with direct combustion routes of macroalgae for electricity or combined heat and power production is the fouling of boilers. This setback is as a result of the production of solid inorganic residues due to the high inorganic mineral contents of macroalgae which have a detrimental effect on the overall efficiency of combustion [26]. Notably, macroalgae have been found to have a higher alkali index (i.e., the amount of alkali metal oxide in fuel per unit of energy expressed as kg alkali GJ-1) of 16-82 kg alkali GJ-1 which is highly above the recommended level (0.17 kg alkali GJ-1) to avoid potential fouling of combustion/boiler systems. It is therefore difficult to envisage the use of macroalgae as alternate viable feedstock in combustion systems without failure if pretreatment to reduce the ash-generating components (inorganic minerals) is not done [16]. Solvent extraction and/or washing pretreatment has been found to significantly affect the concentration of inorganic species which are mostly group I and II metals that are prevalent in marine macroalgae [27]. Removal of such inorganics from macroalgae would enhance the process of the conversion of such biomass into bioenergy whiles the recovered inorganics may be used as a valuable bioproducts or chemical. It is therefore important to understand which extraction conditions and the state of raw seaweed, either fresh or dried, are better for the removal of inorganics to make substrates more suitable for bioenergy production. A broad spectrum of solid-liquid extraction techniques including Soxhlet extraction, maceration, percolation,

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turbo-extraction (high-speed mixing), sonication, supercritical fluid extraction, microwaveassisted extraction, and pressurized solvent extraction could be used. However, some of the solid-liquid extraction methods are often time-consuming and employ relatively large quantities of polluting solvents [28]. The selection of a particular method is an important step to the product quality, yield and a key factor of the production cost for the extraction of macroalgae [29]. According to our knowledge, no study has reported on or compared the inorganic minerals recovery from fresh and dried macroalgae feedstocks. The objective of this study was to investigate the effects of extraction conditions including solid to liquid ratio, temperature, and time as well as solvent and additive concentrations on the inorganic and organic minerals extraction efficiency from fresh and dried alga Saccharina japonica. The ratio of inorganic to organic extraction efficiency was assessed to determine appropriate extraction conditions and to obtain biomass resources with higher quality for the production of bioenergy from macroalgae. 2. Materials and methods

2.1. Materials EtOH (Ethanol, 98%) and sodium chloride (99.5%) were purchased from Samchun Pure Chemicals Co. (Pyeongtaek, South Korea) whilst glacial AA (acetic acid, 99.5%) was purchased from J.T. Baker (Phillipsburg, USA). Fresh seaweed, S. japonica, was purchased from a local market in Daejeon, South Korea. 2.2. Pre-treatment and storage of Saccharina japonica

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Blades of fresh S. japonica biomass were washed 3-4 times with running tap water and finally rinsed with deionized water to remove debris. They were then kept in plastic bags and preserved in a refrigerator at 4 oC to prevent or inhibit possible microbial degradation of alginate as observed by Moen [30]. Prior to extraction, some of the preserved fresh feedstock (FFS) was shredded into 1 x 1 cm pieces. For dried feedstock (DFS) extraction, some portions of fresh biomass were shredded into 2 x 2 cm and dried in an oven for 18 h at 105 oC to remove moisture after which the sizes reduced to the similar size of FFS.

2.3. Solvent extraction Extraction of resources from FFS and DFS materials was achieved using a batch system of extraction. Supplementary Fig. S1 shows the block flow diagram of the entire extraction process. Five grams of FFS or DFS materials were weighed into separate 75 ml vials. To investigate the effect of solid to liquid ratio (S/L) on extraction efficiency, the amount of seaweed biomass to extraction solvent were measured in the aspects of 1:4, 1:6, 1:8, 1:10 and 1:12 ratios (w/v). The DFS has the tendency to absorb some amount of water to swell, hence, 1:4 was selected as the minimum S/L ratio since that could make the separation of supernatant solutions from solids feasible. Into separate vials with FFS and DFS, deionized water was added according to each S/L ratio. Into another separate set of samples, EtOH was used as the extraction solvent. The samples were macerated in a shaking incubator (Model VS8480SF, Vision Scientific, Korea) at 150 rpm for 1 h at 30 oC. Separation of supernatant solutions and solid residues were achieved by vacuum filtration with an electric aspirator (Model VE-11, Lab Companion, Korea) and a filtration apparatus (Nalgene Waltham, USA)

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through 0.45 μm Whatman filter paper. To determine the mass of dried extracts in the filtrates after extraction, 5 ml portions of each filtrate were dried using aluminum dishes in an oven at 105 oC for 2 h. The dried extracts were cooled in a desiccator for 10 min, and weighing was done without delay. The FFS residues (FR) and the DFS residues (DR) were dried in an oven at 105 oC for 12 h to remove moisture and the inorganic content also referred to as ash content [31] were determined by combusting in a muffle furnace at 600 oC according to ASTM E1755-01 [32]. To investigate the effect of agitation time for extraction, the following time intervals were set; 0.5, 1, 2, 3, 6 and 12 h. Varying the extraction time, the S/L ratio was fixed at 1:6 with 30 oC at a mixing speed of 150 rpm. The mass of dried extracts in the filtrates were determined after both fresh and dried biomass extractions. The effect of extraction temperature was determined by the following conditions of 20, 30, 40, 50 and 60 oC with 1: 6 S/L ratio, 1 h mixing time at 150 rpm. EtOH concentrations of 0, 10, 20, 50 and 100 v/v% as well as AA additive concentrations of 0, 1, 2, 5 and 10 v/v% were also assessed on the extraction efficiency of the two biomass types with 1: 6 S/L, 1 h mixing time, 30 oC extraction temperature at 150 rpm. To determine the effect of salinity of water on extraction efficiency, saline solutions of 0, 1.5, 2.5, 3.5, 4.5, 5.5 and 6.5% NaCl were used for FFS extraction at conditions of 1: 6 S/L, 1 h mixing time, 30 oC at 150 rpm.

2.4. Physico–chemical analyses The elemental compositions of CHONS of FFS, FR, DFS, and DR were analyzed using Elemental analyzer (Model EA1108, Fisons, Italy). The percentage of oxygen was calculated

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from the result of the summation of percentages of carbon, hydrogen, nitrogen, and sulfur, subtracted from 100 according to ASTM D3176 [33]; [34]. The analysis of metal concentrations in feedstocks and residues after extraction was done using Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES, Model 5300DV, Perkin Elmer, USA). The morphology of samples was observed by Scanning Electron Microscope (SEM, Model JSM-6390, Jeol, USA).

2.5. Data calculation The extraction efficiency (Etot, %) for total mass, the extraction efficiency for inorganic materials represented by the mass of ash (Einorg, %) and the extraction efficiency for organic materials represented by the mass of organics (Eorg, %) can be obtained from experimental data by the following equations:

𝐷𝑊

𝐸𝑡𝑜𝑡 (%) = (𝐷𝑊 𝐸 ) X 100

(1)

𝐹𝑆

𝐸𝑖𝑛𝑜𝑟𝑔 (%) = (1 −

𝑓𝑖𝑛𝑜𝑟𝑔,𝑅 ∙𝑓𝑅 𝑓𝑖𝑛𝑜𝑟𝑔,𝐹𝑆

𝑓𝑜𝑟𝑔,𝐸 ∙𝑓𝐸

𝐸𝑜𝑟𝑔 (%) = (

𝑓𝑜𝑟𝑔,𝐹𝑆

) X 100

(2)

) X 100

(3)

where DWFS (g) is the dry weight of feedstock before extraction, DWE (g) is the dry weight of extract after extraction, finorg,FS (-) is the fraction of ash in feedstock, forg,FS (-) is the fraction

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of organics in feedstock, finorg,R (-) is the fraction of ash in residue after extraction, forg,E (-) is the fraction of organics in extract after extraction, fR (-) is the mass fraction of residue after extraction and fE (-) is the mass fraction of extract after extraction. The ratio of the extraction efficiency of inorganic materials to that of organic materials after extraction (rE) was determined by the following equation:

𝑟𝐸 =

𝐸𝑖𝑛𝑜𝑟𝑔

(4)

𝐸𝑜𝑟𝑔

2.6. Statistical analysis Each treatment during extraction was replicated three times. The results were expressed as the mean value ± the standard deviation. All the data were subjected to statistical analyses using SPSS 16.0. The main effect of each extraction parameter on the Etot was subjected to analysis of variance and the Tukey-Kramer’s multiple comparison tests using a One-Way ANOVA procedure. This was used to assess the difference between and the significance of yields. Mean values were considered significantly different at p < 0.05.

3. Results and discussion

3.1. General property of the extraction A general study conducted on the extraction showed that the extraction efficiency for FFS-water extraction increased from time 0 h to an optimum time of 2 h but did not vary

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significantly from 2-12 h. There was no significant difference in efficiency from time 0-12 h for DFS-water extraction (Supplementary Fig. S2a). Since much of the minerals were deposited on the surface of materials during drying and an equal amount of water was used, notwithstanding the fact that such minerals have high solubility in water, equilibrium for material transport across the cell wall of the material was quickly attained almost at 0 h. Thus, the osmotic pressure between the inside and outside of the biomass cell arrived at equilibrium quickly. A similar effect was realized by Sun et al. [35], during the classical extraction of alltrans-β-carotene from citrus peels using various solvents. For FFS-water extraction, efficiency increased up to 40 oC (Supplementary Fig. S3a), however, an optimum efficiency was obtained at 30 oC for DFS-water extraction. With FFS-EtOH extraction, an optimum yield occurred at 0.5 h as shown in Supplementary Fig. S4a. Regardless of the extent of the extraction time, very low efficiency values were obtained from 0-12 h for DFS-EtOH extraction due to the fact that the dried minerals have very low solubility in pure EtOH. There was not much significant difference in efficiency in the range of 20-60 oC in FFS-EtOH extraction (Supplementary Fig. S5a). The effect with DFS-EtOH extraction was entirely low due to the solubility effect of EtOH on the dried minerals in DFS.

3.2. Physico – chemical characterization of feedstock and various residues Comparing the ash amount in the remaining residues after extraction as shown in Table 1, it is remarkable that DFS-EtOH extraction had the lowest extraction efficiency since the ash content in feedstock prior to extraction (51.14 ± 1.22%) was almost same as that in DR-

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EtOH extraction (49.25 ± 0.11%). Since the FFS-water and EtOH extractions were much efficient, the remaining inorganics in residues reported as ash were relatively lower as compared to DFS-water and EtOH extractions. It could also be inferred that due to the inefficiency of extraction with DFS using EtOH, the percentage carbon in feedstock prior to extraction and DR-EtOH were the same according to the elemental analysis (CHONS). It was observed that excessive dehydration of water (which forms about 90 % of the wet mass of seaweed) by EtOH was the reason for the mineral extraction during the FFS-EtOH extraction. The dehydration process caused denaturation of the cell wall, thus they were raptured and the remaining unextracted crystals in the FR after extraction formed lumps as revealed by the SEM image in Fig. 1 unlike the layered crystals found after FFS-water extraction. The drying of S. japonica feedstock prior to extraction caused a decrease in volume and weight reduction of biomass. The long chain gummy-like structures as found in the matrix of FFS were broken into brick-like crystalline structures (Fig. 1) due to the excessive evaporation of water. Consequently, there was much mineral salts deposition on the surface of materials after the drying process. These mineral salts which are mostly alginic salts of sodium and potassium are capable of absorbing water quickly. In its extracted form it can absorb an amount of water which is equivalent to 200-300 times of its own weight [36]. The SEM-EDS and the ICP-OES (Supplementary Fig. S6) results revealed that most of the elements found in the FFS and DFS before and after extraction were sodium and potassium rich. The elemental analysis did not indicate the presence of some elements like nickel (Ni), strontium (Sr), lithium (Li), bismuth (Bi), indium (In), molybdenum (Mo), etc., but for

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magnesium (which were 11.5 and 25.1 mg/g in the FFS and DFS respectively). Such unidentified elements could be below the detection limit (0.0001 mg/g) of the measuring instrument. In addition to Mg, higher amounts of Na and K and other elements such as Ca, S, and P were also found which could serve as a valuable resource such as its usage in fertilizer production. 3.3. Effect of solid to liquid ratio-water extraction During the water extraction of FFS, the Etot increased with increasing the S/L from 1:4 (Etot = 39.90 ± 0.83%) to 1:8 (Etot = 51.33 ± 1.33%) (p ˂ 0.05) as shown in Fig. 2a. The ANOVA statistical analysis indicated that there was no significant difference in Etot when the S/L were further increased from 1:8 to 1:12 since p (0.07) > α (0.05). Vongsangnak et al. [17], also found that a larger solvent volume did not necessarily increase the saponin yield from cultured cells of Panax notoginseng by microwave extraction. During the initial phase of extraction, water entered the cell of the FFS by osmosis where water molecules were transported across the highly permeable cell wall rapidly into the inner matrix of the material. This was based on the relatively higher concentration of mineral salts inside the material than in the extraction medium outside. As the pressure within the cell wall increased, reverse osmosis occurred and there was rapid diffusion of dissolved substances into the extraction medium. Although significant denaturation of the seaweed cell wall was not observed before and after FFS-water extraction, permeability increased. The extract-water mixture was forced to flow out of the cell by the sudden change of the internal cell pressure by diffusion which could be considered the rate-determining process. Moreover, the salinity of the extraction water was found to play a notable role. It could be inferred from Supplementary Fig. S7 that

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as the salinity of extraction water increased from 0-6.5% NaCl, the Etot for FFS-saline water extraction reduced drastically. Beyond the average absolute salinity of seawater which is 3.5% of dissolved salts [37], the negative effect on Etot was more pronounced. Therefore, the higher the salinity of the extraction water, the lower the rate of osmosis and subsequently, the lower the rate of diffusion of dissolved extracts. The Einorg as expected were also found to decrease with a corresponding decrease in Etot as salinity of extraction water was increased. In the case of DFS, the Etot rather decreased as the S/L was varied from 1:4 (Etot = 69.77 ± 2.56%) to 1:12 (Etot = 49.32 ± 2.52%) (Fig. 2a). It is notable that the effect of S/L ratio for DFS was opposite to that for FFS. At low S/L ratio, the Etot was higher in DFS than in FFS, whereas the extraction of FFS began to be higher than that of DFS from 1:10. This suggests that the larger amount of water rather inhibits the extraction of materials from dried seaweed. Generally, Einorg for each respective material type at the various S/L was higher than Etot. At 1:4 S/L, Einorg and the fraction of inorganics in extract based on the mass fraction of extract after extraction (finorg,E) were both slightly higher for the DFS-water extraction than for FFS. However, from 1:6-1:12 S/L Einorg and finorg,E increased with increasing the water amount whilst that of the DFS-water extraction was vice versa. The DFS absorbed much of the water meant for extraction rapidly and eventually swelled to become like FFS. However, most of the mineral salts on the material surface were dissolved by the water and remained in solution at high concentrations when the extraction water was less. As the amount of extraction water increased in the variation of S/L ratio, most dissolved surface mineral salts got diluted and absorption through the cell wall occurred by osmosis. Thus, the extraction efficiency decreased with increasing the water amount.

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3.4. Effect of solid to liquid ratio-ethanol extraction Fig. 3a shows that, during the extraction of FFS with EtOH, there was no significant difference in Etot from 1:4 to 1:6, as well as from 1:8 to 1:12 S/L. However, the absolute difference (3.21) of Etot between 1:6 and 1:8 S/L (respectively 43.69 and 46.90%) was greater than the critical range value (1.44) during the multiple comparison test as shown in Table S1a in the supplementary information. Thus, 1:8 S/L could be considered the optimum condition. The Einorg which were higher than Etot just as with the effect of the FFS and DFSwater extraction, were almost same at the different S/L ratios. Since Einorg from using EtOH as a solvent were similar to 1:8 to 1:12 S/L conditions when water was used as a solvent, it would be cost effective to use water instead of EtOH for extraction. However, the formation of a gel film from the highly viscous supernatant solution when water was used, blocking the filter membrane is prevented by the use of EtOH. The entire Etot of DFS-EtOH extraction was relatively low (1.01 ± 0.10-1.83 ± 0.03%). This indicates that the gelatinous materials in kelp seaweeds which are usually monovalent salts of alginic acid [38], mostly sodium and potassium alginates, when dehydrated, has very low solubility in ethanol. The 1.01 ± 0.10-1.83 ± 0.03% Etot was realized due to some bound moisture of 9.35% according to the proximate analysis of dried materials. Etot for DFS extraction with EtOH was very small, but that for FFS was not. This fact means that some inorganic and organic materials dissolved in water present in FFS were extracted together with water.

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3.5. Effect of EtOH concentration Considering the formation of gel film that continually blocked the filter membrane when water was used as a solvent and also the cost of EtOH for extraction, an EtOH-water mixture was investigated for extraction. From Fig. 4a, the effect of solvent concentration was such that, as the concentration of EtOH increased the Etot decreased. There was no significant difference in Etot values for 10 and 20% EtOH (47.77 and 47.30% respectively) as well as 50 and 100% EtOH (43.84 and 45.28%) as indicated in Table S1b in the supplementary information. The absolute difference between 10 and 20% EtOH (0.46) and 10 and 100% EtOH (0.95) were less than the critical range value (3.02) unlike between 10 and 50% (3.93). Since the sap material from FFS is very slimy, filtration of the supernatant solution was difficult with 0% EtOH because of the formation of a gel film that blocked the pores of the filter membrane. However, as EtOH concentration was increased, separation of supernatant solution from residues by filtration was much easy. A minimum concentration of EtOH (10%) was adequate for higher recovery efficiencies. Hence 10% EtOH was the optimum concentration since its effect was similar to 20 and 100% but higher than 50 % EtOH concentration. The Etot slightly decreased in the case of DFS as the EtOH concentration was increased from 0 to 50%, however, the rapid swelling of the DFS by water absorption as observed in section 3.3 was prevented. At 100% EtOH concentration the Etot was very low as reported earlier due to the one reason that the dried minerals do not dissolve in EtOH. The use of EtOH at an optimum concentration helped to overcome the tendency of the dried materials to absorb water rapidly for swelling which affected the Etot negatively. Moreover, the highest Einorg for both FFS and DFS (79.52 and 80.96% respectively) were realized with

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10% EtOH concentration. Hence the optimum EtOH concentration was selected as 10%. Although the Etot for DFS-extraction were quite higher than for FFS from 0-50% EtOH concentrations, the Einorg as well as the finorg,E (shown in Fig. 4) for both feedstock types within such concentration ranges were very similar. At 10% EtOH which was the optimum concentration for both feedstock types, the forg,E for DFS (0.32 ± 0.01) was higher than in the FFS (0.15 ± 0.01). Since the target materials for extraction are inorganics so as to make the remaining residues devoid of much inorganics and a suitable substrate or feedstock for bioenergy production, FFS-extraction was more feasible.

3.6. Effect of AA concentration When AA was added, the extraction efficiency increased significantly from 0-2% AA during the FFS-extraction. The Etot obtained with the use of 2% AA (62.02%) was higher than for 0 and 1% AA concentrations (42.13 and 57.95%). There was no significant difference when the AA concentration was increased from 2-10% AA as shown in Fig. 5a and from the multiple comparison test in Table S1b in the supplementary information. Therefore, 2% AA was adequate for the FFS extraction. For the DFS-extraction, there was no significant difference in Etot when the concentration was varied from 0-10% AA (p ˃ 0.05). However, it was observed that the introduction of AA additive prevented the rapid swelling by the absorption of water molecules by the already dried biomass. In the case of DFS extraction with water without any AA solvent additive, there was such rapid swelling of biomass since the dried alginic salts sap materials have a higher tendency of absorbing a relatively higher quantity of water for rehydration. Thus with AA additive, there was no decrease in extraction

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efficiency unlike for DFS water extraction. An optimum Einorg for FFS (86.32 ± 0.10%) with finorg,E (0.44 ± 0.0005) was obtained with 2% AA whiles for DFS, 1% AA for DFS resulted in an optimum Einorg (79.55 ± 1.90%) with finorg,E (0.41 ± 0.01). Hence, relatively lower concentrations of AA are necessary for the effective recovery of inorganic materials from both feedstocks.

3.7. Ratio of inorganic and organic extraction efficiency The ratio (rE) of comparison of Einorg to Eorg as a result of S/L as well as EtOH and AA concentrations during extraction are illustrated in Fig. 6. Since rE compares Einorg to Eorg, higher rE values above 1 indicates a more successful removal of inorganic materials from a particular feedstock other than organic materials needed as feed for most biorefineries. From Fig. 6, Einorg was much higher than Eorg in FFS extractions with pure water compared to that in DFS extractions. The rE decreased with increasing S/L ratio from 1:4 to 1:12. At 1:4 of S/L ratio with FFS, rE was 13.80, which means that inorganic materials were effectively extracted much more than organic materials at such a low water requirement. The rE for DFS-water extraction, unlike for FFS, were just slightly above 1 at all S/L ratios. This means that for FFS, inorganic extractions were more efficient than DFS. For FFS-EtOH extraction, the rE decreased with increasing S/L ratio (Fig. 6a), similar to the trend of FFS-water extraction. Also, at 1:4 S/L ratio the highest rE (17.05 ± 3.94) was obtained for FFS-EtOH indicating a lower EtOH amount for effective inorganic materials removal. Notably, for DFS-EtOH extraction at all S/L ratios, rE was denoted NA since DFSEtOH extractions were not efficient due to the very low solubility of minerals with 100%

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EtOH. From Fig. 6b, rE at 1:6 S/L ratio increased with increasing EtOH concentration from 0 to 100%, with the effect at 10 and 20% EtOH being similar during extraction with FFS. The values of rE for DFS extraction were just slightly above 1 with 10 and 20% EtOH having the highest rE (~ 2). The use of AA additive did not increase the values of rE as the concentrations were increased from 0 to 10% for both FFS and DFS. At all concentrations, FFS-AA additive extractions resulted in slightly better rE values than for DFS-AA additive extractions. Previous investigations have shown that the presence of higher inorganic compounds mostly in marine biomass sources often has the tendency to inhibit the successful conversion of such resources into bioenergy and other bioproducts [23], [39], [40]. Kim et al. [39] found that although the yields of oil, gas, and char are mostly functions of process conditions such as temperature and time, a large amount of inorganic compounds in S. japonica resulted in higher char yields during pyrolysis. Also, Choi et al. [23] found that sulfuric acid pretreatment of S. japonica significantly reduced the presence of catalytically active inorganic minerals but did not significantly affect the properties of pyrolysis oil compared to a non-treated kelp pyrolysis oil. Rather, the non-treated kelp produced larger chunks of biochar which affected the continuous operation of the fluidization bed reactor whereas the acid pretreated biomass offered a more convenient continuous process since the later did not result in aggregated large particles of char. Moreover, a study by Ly et al. [40] on the pyrolysis of Cladophora socialis alga in a microtubing reactor showed that the higher presence of inorganic compounds in the feedstock also resulted in higher char yields and degraded the quality of the product bio-oil. Therefore, an effective extraction of the characteristic high inorganic minerals from

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macroalgae, either fresh or dried, would provide a better biomass resource for biorefinery. Henceforth it is notable that, rE from the pretreatment of such inorganic-rich macroalgae feedstocks has a very important significance in most biorefinery operations where the target component of algal materials is the organics.

3.8. Overall assessment The overall assessment of mineral extraction from fresh and dried kelp seaweed biomass based on the efficiency of recovery of inorganic minerals is summarized in Table 2. Purity of organic and inorganic portions remaining after extraction (finorg,R and forg,R respectively) expressed in percentages was estimated after the analysis of ash content of residues. After FFS extraction with 10% EtOH (optimum concentration), the potential of finorg,R in the remaining residue was 20.05 ± 0.01% whiles forg,R was 79.95 ± 0.29. With 2% AA additive concentration, finorg,R was 18.43 ± 0.05% whiles forg,R was 81.57 ± 0.44%. In the case of DFS extraction, the estimated potentials were; finorg,R = 24.99 ± 0.16% and forg,R = 75.01 ± 1.34% for 10% EtOH concentration with finorg,R = 30.69 ± 0.97% and forg,R = 69.31 ± 2.30% for 1% AA additive concentration. Since the Einorg in FFS were generally effective and higher than in DFS, the forg,R which is the target substrate or feedstock for bioenergy conversion were higher in FFS residues than in DFS residues. The estimated potential of residues after DFSEtOH extraction showed that 100% EtOH was not suitable for extraction. Though for the FFS – extraction, 2% AA additive concentration resulted in the highest Etot and Einorg, however the rE was relatively lower as compared to other solvent concentrations. The 100% EtOH resulted in the highest rE but with a relatively lower Einorg.

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Also 10% EtOH, resulted in relatively higher Einorg after that of 2% AA and a higher rE after that of 100% EtOH concentration. For the DFS-extraction 1% AA and 100% water resulted in the highest Etot. It is notable that, from 0 to 100% EtOH, 10% EtOH resulted in the highest Einorg . Since the DFS-extraction with 100% EtOH was inefficient, the Etot as well as Einorg were very low. Furthermore, as compared to FFS, the drying of algal biomass is very energy intensive. Typically, a study by Milledge and Heaven [41], on the review of energy extraction from microalgal biomass revealed that to heat water from 20 to 100 oC with subsequent evaporation at atmospheric pressure requires ~ 2.6 MJ kg-1 of energy input. Again, the energy required for the dehydration or drying of the seaweed is higher than the Heating Value of dried seaweed [42], [43]. Hence, there is an increment in the overall process energy input requirement due to drying (to obtain DFS) prior to inorganic mineral extraction. Therefore, considering the extraction efficiency of the target inorganic materials and the additional cost of drying, there would be much value addition after carrying out extraction using FFS over DFS. Considering the rE values and the cost of drying for the DFS, it is economically more efficient to extract resources from fresh kelp seaweed than dried biomass.

4. Conclusions

Minerals extraction from S. japonica revealed that Einorg were much higher than Eorg at all conditions during FFS extraction than in DFS. The rE values greater than 1 signifies

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inorganic materials recovery was more efficient making the organic-rich residue devoid of much inorganics, hence, rendering it more useful as a bioenergy resource. The addition of EtOH and AA at low concentrations (10% EtOH and 1-2% AA) enhanced easy separation of supernatant solution containing inorganic materials from organic-rich residues. This study should be useful for optimization of pretreatment of macroalgae feedstocks, thereby providing a better biomass resource for biorefinery conversions.

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Figure Captions

Fig. 1. SEM images of (a) FFS (b) FR-water extraction (c) FR-ethanol extraction (d) DFS (e) DR-water extraction and (f) DR-ethanol extraction. Fig. 2. Effect of S/L ratio on (a) the extraction efficiency of minerals recovery from FFS and DFS using water (b) the fraction of inorganic minerals in FFS and DFS extracts. Fig. 3. Effect of S/L ratio on (a) the extraction efficiency of minerals recovery from FFS and DFS using ethanol (b) the fraction of inorganic minerals in FFS and DFS extracts. Fig. 4. Effect of EtOH concentration in water on (a) the extraction efficiency of minerals recovery from FFS and DFS (b) the fraction of inorganic minerals in FFS and DFS extracts. Fig. 5. Effect of AA concentration in water on (a) the extraction efficiency of minerals recovery from FFS and DFS (b) the fraction of inorganic minerals in FFS and DFS extracts. Fig. 6. The ratio of Einorg to Eorg as a result of the effect of (a) S/L ratio (b) solvent concentration.

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Fig. 1.

29

(a)

(b) Fig. 2.

30

(a)

(b) Fig. 3.

31

(a)

(b) Fig. 4.

32

(a)

(b) Fig. 5.

33

(a)

(b) Fig. 6.

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Table 1 Elemental analysis and ash content of unextracted S. japonica seaweed feedstock and its residues after extraction. Dried seaweed biomass

Carbon %

Hydrogen %

Oxygen %

Nitrogen %

Sulfur %

††Ash

Feedstock

21.63 ± 0.42

2.92 ± 0.01

29.71 ± 2.10

1.16 ± 0.02

0.55 ± 0.06

51.14 ± 1.22

FR-water extraction

29.07 ± 0.63

4.2 ± 0.11

44.47 ± 0.98

1.69 ± 0.09

0.37 ± 0.01

20.68 ± 2.92

FR-ethanol extraction

24.26 ± 0.55

3.52 ± 0.21

38.49 ± 0.99

1.68 ± 0.05

ND†

17.93 ± 0.13

DR-water extraction

25.23 ± 0.41

3.41 ± 0.03

38.2 ± 1.28

1.52 ± 0.10

ND†

37.19 ± 0.14

DR-ethanol extraction

21.62 ± 0.28

3.07 ± 0.08

28.09 ± 2.23

1.37 ± 0.07

ND†

49.25 ± 0.11

†Not

detected. by ASTM E1755-01[32].

††Measured

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%

Table 2 Overall assessment of minerals recovery from FFS and DFS using water, EtOH and AA. Feedstock

FFS

DFS

Etot, %

Extraction parameters* Einorg, % rE

finorg,R, %

forg,R, %

100 (W)

50.82 ± 0.41

76.88 ± 0.08

3.27 ± 0.10

24.05 ± 0.04

75.95 ± 0.36

10 (EtOH)

47.77 ± 0.28

79.52 ± 0.02

5.48 ± 0.22

20.05 ± 0.01

79.95 ± 0.29

100 (EtOH)

42.21 ± 0.36

74.19 ± 0.004

8.53 ± 0.73

22.84 ± 0.002

77.16 ± 0.36

2 (AA)

62.02 ± 0.49

86.32 ± 0.09

2.36 ± 0.06

18.43 ± 0.05

81.57 ± 0.44

100 (W)

65.79 ± 3.10

72.99 ± 2.36

1.26 ± 0.04

40.38 ± 1.20

59.62 ± 1.90

10 (EtOH)

61.02 ± 1.50

80.96 ± 0.31

2.02 ± 0.13

24.99 ± 0.16

75.01 ± 1.34

100 (EtOH)

1.21 ± 0.62

7.28 ± 0.37

NA

48.00 ± 0.19

52.00 ± 0.20

1 (AA)

65.92 ± 3.27

79.55 ± 1.90

1.55 ± 0.10

30.69 ± 0.97

69.31 ± 2.30

Concentration, %

NA – not applicable. *

The conditions for extraction were 1:6 S/L ratio, 1 h extraction time and 30 oC temperature at 150 rpm mixing speed.

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