Deconstruction of the green alga Ulva rigida in ionic liquids: Closing the mass balance
Graphical abstract
Introduction
The scientific community agrees that when the Earth was formed, its atmosphere did not yet have enough oxygen to support more complex forms of life. Nonetheless, there is evidence that as early as 3700 million years ago a particular type of prokaryotic microorganism containing chlorophylls, known as cyanobacteria, started to perform photosynthesis by taking up organic and inorganic carbon sources to produce energy and oxygen. Consequently, these microorganisms were most likely responsible for creating our oxygen-rich atmosphere. Biologists believe that algae evolved from these microorganisms, as they are also organisms containing chlorophyll capable of performing photosynthesis to produce energy and oxygen [1]. Algae species present in the marine world can be classified into two major groups: microalgae and macroalgae. Microalgae are mainly unicellular microorganisms, while macroalgae are multicellular organisms. Multiple species of both types of algae have a high content of carbohydrates, as well as protein and lipids, all of which could be used in the production of bioethanol, biogas and biodiesel. In fact, Algenol Biofuels, Inc. in the United States is a good example of utilization of genetically modified microalgae for bioethanol production. Algae are also well known to possess a higher growth rate compared to terrestrial biomass. Macroalgae are divided into three big groups: green algae, red algae and brown algae. All of them are characterized by being composed of different kinds of carbohydrate species, but most importantly they completely lack lignin and hemicelulloses, thus diminishing the need for any aggressive preprocessing techniques aiming at deconstruction of the carbohydrate matrix to release fermentable sugars [2], [3], [4], [5], [6].
Green algae are characterized as containing starch applicable as a food resource and polysaccharides containing sulfate esters, mainly composed of arabinose, galactose, rhamnose, xylose and glucuronic acid [7], [8]. Red algae, in turn mainly contain cellulose, glucan and galactan sulfates as polysaccharide species [4], [7], [8]. Ultimately, brown algae mainly contain alginate as one of their main carbohydrate components, but they have fucoidan as a polysaccharide containing sulfated esters, as well as mannitol and laminarin [4], [6], [7], [9], [10].
Ulva rigida (C. Agardh 1823) is a type of seaweed belonging to the group of green algae tending to grow near submerged marine rocks in many seas and oceans across the world [11]. The main constituents of U. rigida are carbohydrates, protein, ash and lipids. Further, starch and cellulose are among the carbohydrate species contained in U. rigida. Zemke-White & Clements [12] reported 3.5 wt.% of starch in U. rigida and Roesijadi et al. [6] and Percival [8] have reported the content of cellulose to be very low.
On the other hand, ulvan is also found in the U. rigida species, a polysaccharide containing rhamnose, xylose, glucuronic acid and sulfate esters. Fig. 1 depictures the chemical structure of ulvan, which shows great complexity by repetition of a number of disaccharides within different species. The most common repeating units are ulvanobiouronic acid 3 sulfate type A (As3) and B (Bs3) shown in Fig. 1A and B, respectively [13]. As3 is composed by the dimer β-d-glucuronosyluronic acid-(1 → 4)-l-rhamnose-3-sulfate, whereas Bs3 is formed by the dimer α-l-iduronosyluronic acid-(1 → 4)-α-l-rhamnose-3-sulfate. In turn Fig. 1C depicts ulvanobiose 3-sulfate (Us3) dimer composed by a xylose unit replacing uronic acids, which can also contain a sulfate group in positions C-2 or C-3 as shown in Fig. 1D. Nevertheless, its presence is reported to be fewer compared to ulvanobiouronic acid 3 sulfates. Oligosaccharides containing residues of glucuronic acid in As3 dimers can also be found in ulvan (see Fig. 1E). Further the presence of galactan sulfates as minor components has also been reported in some Ulva species [13], [14], [15], [16].
Ionic liquids (IL) are known to be able to dissolve polysaccharides and carbohydrates such as cellulose as well as to disrupt the complex linkages of pristine biomass [17], [18]. In fact, dissolution of algae biomass in ionic liquids has been reported earlier for different types of algae. Species such as Sargassum fulbellum, Laminaria japonica and Undaria pinnatifida in the group of brown algae were processed in 1-n-butyl-3-methylimidazolium chloride ([BMIM+][Cl−]) containing different types of acids, whereupon yields approaching 99 wt.% of total reducing sugars were reported, at temperatures within the range of 100–150 °C and processing times up to 250 min. Further, it was claimed that, when treating algae at temperatures below 120 °C, the hydrolyzate purity was greater than 95 wt.%, albeit decreasing when exposure time increased. The authors claimed that carbohydrate hydrolysis in ionic liquid mediated systems rendered better results when compared to dilute sulfuric acid treatments in an autoclave [19]. Saccharina japonica, a brown alga mainly cultivated in the coasts of China, South Korea and Japan, was also subject to processing with different kinds of ionic liquids such as 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM+][BF4−]), n-butyl-4-methylpyridinium tetrafluoroborate ([BMPy+][BF4−]) and n-methylmorpholinium hydrogensulfate ([NMM+][HSO4−]) containing zinc chloride (ZnCl2). As a result, the authors reported hydrolyzates with reducing sugars concentrations of 6.2, 6.4 and 6.0 g/L, respectively [20]. Besides the ionic liquid mediating process, S. japonica and L. japonica were exposed to low concentration, diluted sulfuric acid treatments, whereby subsequent simultaneous saccharification and fermentation (SSF; only for S. japonica) was performed in order to produce bioethanol. The acid concentration was varied within the range of 0.02–0.14 wt.%, at temperatures ranging from 150 to 180 °C and reaction times from 5 to 20 min. The enzymatic digestibility for S. japonica and L. japonica (enzymatic hydrolysis) was enhanced compared to untreated algae, whereby yields of 84.0 and 83.4 wt.% were reported, respectively [21], [22].
Earlier reported ionic liquid preprocessing of green algae has mainly been focused on Chlorella biomass, a unicellular microorganism within the group of microalgae. Chlorella biomass is characterized as constituting high amounts of lipids, proteins and carbohydrates. Depending on the species, the typical values are about 20 wt.% fat, 45 wt.% protein and 20 wt.% carbohydrates [23]. Chlorella biomass treated with 1-ethyl-3-methylimidazolium chloride ([EMIM+][Cl−]) at 105 °C for 3 h, coupled to a subsequent treatment with concentrated 7 wt.% hydrochloric acid, relative to biomass content, at 105 °C for 3 h, resulted in about a 90 wt.% yield of sugars [24]. Choi et al. [25] treated Chlorella biomass with 1-ethyl-3-methylimidazolium acetate ([EMIM+][OAc−]) at 110 °C for 2 h, which resulted in a lipid extraction of 219 mg/g of cells. Chlorella biomass was also treated with 2 wt.% hydrochloric acid and 2.5 wt.% magnesium chloride (MgCl2) at 180 °C for 10 min, resulting in about an 83 wt.% yield of sugars [26]. Interestingly, Lovejoy et al. reported a series of ionic liquids capable of extracting isopropenoids contained in the cellular wall of the green microalga Botryococcus braunii. The treatment reported minimal cellular death and allowed the recovery of the extracted isopropenoids via distillation [27]. Unlike the microalgae, the green macroalga U. rigida has only been characterized in terms of its protein, lipids and carbohydrate content so far, and practically no detailed analysis of its depolymerized and deconstructed products after processing was published to date. Nevertheless, there have been efforts for carbohydrate dissolution using aqueous systems that have accomplished ulvan extraction to a partial extent [28], [29], [30].
In this work, green alga U. rigida was processed in ionic liquids, whereupon the goal was to isolate carbohydrates that can be used in the production of platform chemicals or biofuels like ethanol. Consequently, the selection of the solvents was made on the basis of their carbohydrate dissolution potential. The first IL tested was DBU–MEA–SO2 (1,8-diazabicyclo-[5.4.0]-undec-7-ene, monoethanolamine, sulfur dioxide) the so called switchable ionic liquid (SIL). The term switchable stands for the ability of this type of solvent to be back switched from ionic form to a mix of molecular liquids by bubbling an inert gas (such as nitrogen) to obtain the starting materials (in this case superbase and alkanol amine). This IL has been successfully utilized in e.g. solubilization of lignin and hemicelluloses [31]. The second IL used was 1,1,3,3-tetramethylguanidine (TMG) propionate ([TMGH+][CO2Et−]), known as a distillable ionic liquid (DIL), capable of being distillated at temperatures around 130 °C under relatively high vacuum. This IL has been shown to successfully dissolve microcrystalline cellulose (MCC) with dissolution times as short as 10 min [32]. The third IL used was a protonated 1,8-diazabicyclo-[5.4.0]-undec-7-ene 2,2,3,3,4,4,5,5-octafluoro-1-pentoxide ([HDBU+][5OF−]). This IL was selected because of its very low viscosity (LVIL) which allows for better mixing and improved mass transfer compared to high viscosity ILs. Further, the IL pertains more hydrophobic properties, since its anion is a bulky fluorous, organic molecule which facilitates separation from water. Notwithstanding, this IL has not yet been reported in the literature as a biomass pretreatment or dissolution solvent. Fig. 2 depicts the chemical structure of the ionic liquids utilized in this work. Deconstruction of algae biomass in terms of carbohydrate, proteins and ash in order to determine the mass balance of the process was also another target of this work. No earlier reports on U. rigida processing in ionic liquids were found in open literature and the product mix obtained was comprehensively characterized.
Section snippets
Materials
U. rigida (C. Aghard 1823 Chlorophyta, Ulvaceae) was kindly donated by Prof. Mario Edding from the Research and Technological Center in Applied Phycology – CIDTA – Northern Catholic University, Chile. The algae were collected in 2009 from submerged marine rocks located in La Herradura de Guayacán Bay, in the city of Coquimbo, Región de Coquimbo in Northern Chile. Algae were dispersed on plastic carpets and left to air dry for 48 h avoiding contact with sunlight. As the next step, algae were
Proximate composition of the fresh alga
According to proximate analysis, Ulva rigida contains high amounts of carbohydrates, i.e. 50.1 wt.%. This value is in accordance with earlier reported values [39]. In terms of its potential nutritional value, special attention should be paid to the high protein content of 20.6 wt.%. Consequently, Ulva rigida species are used as a source of food in some regions across Asia because of its high carbohydrate and protein content [40], [41]. This substantial amount of carbohydrates also motivates its
Conclusions
Hereby we report a mild ionic liquid mediated processing to facilitate Ulva rigida biomass deconstruction with extensive characterization of the phenomena occurring. It was demonstrated that TMG propionate ionic liquid is a potent candidate for removal of carbohydrate from the biomass matrix since 67 wt.% of the total carbohydrates was dissolved. The use of DBU–MEA–SO2 SIL and [DBUH+][5OF−] didn’t give as good results as the use of [TMGH+][EtCO2-] and, only approximately 25 wt.% of the
Acknowledgments
This work is a part of the activities of the Johan Gadolin Process Chemistry Centre (PCC), a Centre of Excellence financed by Åbo Akademi University. The Academy of Finland – AKA – (Grant number 268937) and the National Commission for Scientific and Technologic Research of the Government of Chile – CONICYT – (Project AKA-ERNC 0009) are gratefully acknowledged for funding this project. In Sweden, the Bio4energy program, Kempe Foundations (Kempe Stiftelserna) and Wallenberg Wood Science Center
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