Trang Q.
To
*a,
Kerryn
Procter
a,
Blake A.
Simmons
b,
Suresh
Subashchandrabose
c and
Rob
Atkin
ad
aPriority Research Centre for Advanced Fluids and Interfaces, Newcastle Institute for Energy and Resources, Australia. E-mail: Trang.To@newcastle.edu.au
bLawrence Berkeley National Laboratory, Joint BioEnergy Institute, Berkeley, CA 94720, USA
cGlobal Centre for Environmental Remediation, Faculty of Science, University of Newcastle, Callaghan, New South Wales 2308, Australia
dSchool of Molecular Sciences, The University of Western Australia, WA 6009, Australia
First published on 17th May 2017
Biomass based biofuels are already an important energy source, and will increasingly be so in the future as the need for renewable energy rises. Due to their fast multiplication rates, algae can provide a sustainable supply of biomass, and are attractive because they do not compete with food crops for habitat. Here we show that biomass derived from Chlorella vulgaris and Spirulina platensis can be pretreated with low cost choline amino acid based ionic liquids to effectively yield lipids (30.6% and 51% total lipids) and sugars (71% and 26% total sugars). The ionic liquids dissolve the lipids, leaving behind a carbohydrate rich solid. The lipids were extracted with hexane, and the solid was subjected to enzyme hydrolysis to release fermentable sugars. These results open new pathways towards the dual production of biodiesel and bioethanol from algae, using low cost ionic liquids.
The most popular method to extract lipids from microalgae is Soxhlet extraction using hexane,10 but this approach has several disadvantages in terms of commercial viability. First, the cell walls of microalgae are made up of a highly complex matrix of polysaccharides intercalated with proteins,11–13 which has a high chemical resistance to non-polar solvents. Second, hexane is incapable of extracting lipids stored in lipid droplets, as it cannot cross the (protein bound) polar phospholipid-membrane. On the other hand, polar solvents such as methanol/chloroform cross the phospholipid barrier2 by diffusion and extract these lipids (the Bligh & Dyer method14). Physical pretreatment to break open the cells (such as osmotic shock,15 blending, microwave or laser16) followed by the extraction of lipids with solvents has been investigated, but these physical pretreatment steps, together with the need for dry algal biomass in the extraction, result in energy intensive processes that are not commercially feasible.2,3,17
Chemical pretreatment offers a potential alternative that may be commercially viable. For example, a biphasic system of acidic solution and hexane in a bioreactor (155 °C) facilitates the recovery of up to 97% of lipids and the release of 90% glucose from Chlorella and Scenedesmus.18 Certain ionic liquids (ILs), which consist entirely of cations and anions,19–21 are interesting candidates for the pretreatment step due to their favourable physical properties. ILs are tuneable, have the capacity to dissolve a wide range of materials, and low toxicity variants are known.20,21 ILs have attracted research interest for the pretreatment of lignocellulosic biomass for enhanced bioethanol production.22,23 Certain ILs selectively dissolve the lignin component of the lignocellulosic biomass, leaving behind a cellulose-rich residue easily digested by enzymes to release fermentable sugars.
The use of ILs for the pretreatment of algal biomass for lipid extraction has been investigated previously.24–29 ILs work by dissolving the lipids, which separate out of the liquor on addition of an antisolvent such as methanol, which are then recovered using hexane. However, improvement is needed in several areas.24 Most of the ILs employed in these studies are imidazolium- and pyridinium-based, which are ‘classic’ ILs but are expensive and toxic. Only two studies so far have used the cheaper ammonium- and phosphonium-based ILs.29,30 However, regardless of the cation type employed the algal biomass solid loading was low (between 5 and 10 wt%),31 and only a few studies have attempted to recover other valuable components of the algal biomass, such as carbohydrates and carotenoids.32–34
This study explores the use of a series of cheap and environmentally benign ILs for the pretreatment of microalgae. Made from simple acid–base reactions between two naturally occurring non-toxic chemicals, choline (an ammonium) and amino acids, these ILs are significantly cheaper and less toxic than imidazolium-based ILs. To further reduce the cost, instead of using neat ILs, mixtures of IL-water are utilised. This class of ILs has been shown to perform well for lignocellulosic biomass processing,35 and facilitate a one-pot reaction from biomass to ethanol. Two distinct, representative, microalgae are probed, one from the prokaryotic cyanobacterial group, Spirulina platensis, and the other from the eukaryotic green algae Chlorella vulgaris. The latter species C. vulgaris is extensively studied due to its high lipid content (∼300 mg g−1 dry cell). However, C. vulgaris possesses a robust and complex cell wall, which makes it resistant to chemical attack. In order to examine whether our ILs can penetrate this barrier, two samples of C. vulgaris were investigated, one with an intact cell wall, and one with a cracked cell wall. High solid loading (20 w/v%), moderate temperature (70 °C) and a short reaction time (3 h) were employed and both lipids and carbohydrates were recovered.
0.5 g of the biomass samples was pretreated by dissolving in 2.5 mL of the selected ILs at 70 °C for 3 hours with continuous stirring. Ten mL of water was added and the samples were centrifuged to separate the solid and liquid fractions (6000 rpm, 15 minutes). The pretreated biomass solid was subjected to further washing with water (3 × 10 mL water) to remove residual IL.
The GC program (Shimadzu machine fitted with a Restek Rxi-5Sil MS column) was as follows: 1 μL injection at 8:1 split ratio, inlet temperature of 250 °C; constant flow of 1 mL min−1 helium; oven temperature: 100 °C for 1 min, 25 °C min−1 up to 200 °C and hold for 1 min, 5 °C min−1 up to 250 °C and hold for 7 min (23 min total). For the first run, a full scan MS was carried out to identify the structure and the retention time of the FAMEs present. Selected ion monitoring (SIM) was then performed for enhanced sensitivity and accurate quantification. Calibration curves were created from standards made from a C8–C24 FAME mix (Supelco) and the C13:0ME internal standard. The Supelco mix covers most of the FAMEs present in the algal biomass, except for C16:2 and C16:3; whose quantification had to be done using the responsive factors obtained for C18:2n6 and C18:3n3 (present in the Supelco mix) as recommended in the NREL protocol.
In a GC vial, algal biomass (∼20 mg) was mixed together with 40 μg of tetracycline (4 μL of 10 mg mL−1 solution in EtOH), 30 μg of cycloheximide (3 μL of 10 mg mL−1 solution in water), 0.6 μL of cellulase (from Aspergillus sp., Sigma, equivalent to 25 U per g of biomass), 2 μL of α-amyloglucosidase (from Bacillus licheniformis, Sigma, equivalent to 25 U per g of biomass), 2 μL of α-amylase (from Aspergillus niger, Sigma, equivalent to 25 mg protein per g of biomass) and 970 μL of buffer solution (NaOAc/AcOH, pH 5). The vial was vortexed briefly then left at 50 °C with stirring for 24 h. The D-glucose and D-galactose content were measured using the Accu-check Performa Glucose meter.
TGA results | |||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Chlorella vulgaris (Algomed, intact) | Chlorella vulgaris (Synergy, cell broken) | Spirulina platensis | |||||||||||||||
Raw | [Emim][OAc] | [Ch][ARG] | [Ch][GLY] | [Ch][LYS] | [Ch][PHE] | Raw | [Emim][OAc] | [Ch][ARG] | [Ch][GLY] | [Ch][LYS] | [Ch][PHE] | Raw | [Emim][OAc] | [Ch][ARG] | [Ch][GLY] | [Ch][LYS] | |
Moisture% | 7.2 | 10.0 | 14.9 | 10.1 | 11.8 | 10.1 | 7.9 | 6.0 | 8.1 | 8.0 | 7.6 | 7.6 | 6.2 | 7.2 | 7.8 | 7.8 | 8.9 |
Solid% | 85.7 | 84.6 | 85.1 | 85.3 | 87.9 | 84.7 | 88.4 | 92.7 | 88.8 | 92.0 | 92.0 | 92.4 | 88.7 | 89.6 | 90.2 | 89.5 | 91.1 |
Ash% | 7.1 | 5.4 | 0 | 4.6 | 0.3 | 5.2 | 3.7 | 1.3 | 3.1 | 0 | 0.4 | 0 | 5.1 | 3.2 | 2.0 | 2.7 | 0 |
Solid recovery (%) | 67.4 | 30.4 | 74.5 | 59.6 | 66.9 | 52.9 | 25.0 | 61.3 | 68.4 | 61.0 | 82.3 | 24.1 | 51.8 | 38.8 |
Fig. 1 TGA profiles and their first derivative curves for intact C. vulgaris and its treated counterparts. |
Fig. 2 TGA profiles and their first derivative curves for broken C. vulgaris and its treated counterparts. |
Fig. 3 TGA profiles and their first derivative curves for S. platensis and its treated counterparts. |
There is a consistent reduction of ash in all of the pretreated biomass (see Table 2) samples. Ash is composed of minerals together with silica, which can be dissolved using alkali solution. Hence it is not surprising that some of the ash was lost during the pretreatment, since the IL solutions were all basic.
Fatty acids | Chlorella vulgaris (Algomed, intact) | Chlorella vulgaris (Synergy, cell broken) | Spirulina platensis | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Raw | [Emim][OAc] | [Ch][ARG] | [Ch][GLY] | [Ch][LYS] | [Ch][PHE] | Raw | [Emim][OAc] | [Ch][ARG] | [Ch][GLY] | [Ch][LYS] | [Ch][PHE] | Raw | [Emim][OAc] | [Ch][ARG] | [Ch][GLY] | [Ch][LYS] | |
Fatty acid composition of solid residue (% of total lipids) | |||||||||||||||||
C16:0 | 17.3 | 10.2 | 25.2 | 17.4 | 22.5 | 22.3 | 13.2 | 16.1 | 45.0 | 26.7 | 18.6 | 25.1 | 34.1 | 36.6 | 64.3 | 38.3 | 48.2 |
C16:1 | 3.7 | 4.6 | ND | 4.1 | 5.2 | 6.6 | 2.1 | 3.6 | ND | 2.4 | 4.9 | 2.9 | 8.2 | 9.3 | 0.9 | 5.6 | 4.1 |
C16:2 | 4.8 | 1.3 | ND | 3.1 | 1.6 | 5.4 | 5.3 | 6.2 | ND | 1.8 | 1.0 | 3.5 | ND | ND | ND | ND | ND |
C16:3 | 10.5 | 5.4 | ND | 5.3 | 1.8 | 9.6 | 4.5 | 4.7 | ND | ND | ND | 1.0 | 0.4 | ND | ND | ND | ND |
C18:0 | 1.4 | ND | ND | 2.2 | 2.1 | 1.1 | 2.9 | 3.3 | 4.7 | 4.2 | 1.4 | 3.7 | 0.1 | 0.5 | 0.4 | 0.6 | 1.5 |
C18:1 | 10.3 | 2.9 | ND | 13.2 | 13.3 | 9.4 | 8.8 | 8.3 | 1.2 | 10.0 | 5.0 | 8.2 | ND | 1.2 | ND | 0.7 | 2.7 |
C18:2 | 19.7 | 10.8 | 0.7 | 22.6 | 22.1 | 17.8 | 33.1 | 32.5 | 17.8 | 32.2 | 28.6 | 29.9 | 17.0 | 17.2 | 7.3 | 17.5 | 18.9 |
C18:3 | 28.2 | 11.1 | ND | 25.8 | 21.2 | 22.0 | 25.1 | 22.4 | 1.3 | 10.0 | 9.0 | 11.9 | 21.7 | 18.7 | ND | 10.3 | 7.6 |
C22 | 2.1 | 26.5 | 36.4 | 3.1 | 5.0 | 2.9 | 2.7 | 1.6 | 14.8 | 6.3 | 15.5 | 6.9 | 9.1 | 8.0 | 13.3 | 13.3 | 8.3 |
C22:1 | 2.1 | 27.4 | 37.6 | 3.2 | 5.1 | 3.0 | 2.4 | 1.3 | 15.1 | 6.3 | 16.0 | 6.9 | 9.4 | 8.3 | 13.8 | 13.8 | 8.7 |
Total | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% | 100% |
mg g−1 dry solid | 287.9 | 56.1 | 55.4 | 258.7 | 154.7 | 197.3 | 300.1 | 331.1 | 149.6 | 119.8 | 82.4 | 115.6 | 102.7 | 110.1 | 197.2 | 89.5 | 151.4 |
% total lipids | 13.1 | 5.8 | 67.0 | 32.0 | 45.8 | 58.3 | 12.5 | 24.5 | 18.8 | 23.5 | 88.3 | 46.3 | 45.2 | 57.3 | |||
Lipid extracted from the IL liquor by hexane | |||||||||||||||||
mg g−1 original biomass | 7.1 | 28.8 | 8.0 | 13.3 | 8.7 | 14.7 | 100.7 | 6.8 | 17.3 | 36.0 | 7.4 | 52.5 | 13.7 | 28.0 | |||
% total lipids | 2.5 | 10.0 | 2.8 | 4.6 | 3.0 | 4.9 | 33.6 | 2.3 | 5.8 | 12.0 | 7.2 | 51.1 | 13.3 | 27.3 |
The second step of retrieving the lipids involves extracting them from the IL liquor using hexane. The IL liquors were neutralized with acid, and the lipids were extracted using hexane. Without neutralization, no fatty acids were recovered from the IL liquors. Neutralization allows the fatty acids to convert to their neutral form, which is more soluble in hexane than the anionic form. The purity of the crude lipid extracts was analyzed using GC and was found to vary between 20 and 100%. These values were taken into account to calculate the actual lipid yield. In cells, lipids are stored as triacylglycerols in free form and also in lipid droplets, surrounded by a phospholipid monolayer decorated with proteins. For successful extraction of lipids, pretreatment must break down the cell wall and the cell membrane, as well as release the lipids from any bound proteins. It was found that not all the lipids that were removed from the biomass could be extracted using the hexane. Fig. 4 displays the distribution of lipids after pretreatment. [Ch][ARG] further displays its superiority over the other ILs in disintegrating lipids, as the hexane was able to extract 10.0% (intact C. vulgaris), 33.6% (broken C. vulgaris) and 51.1% (S. platensis) of the total lipids (Table 1 and Fig. 4, orange bars). It is also evident that the broken form of C. vulgaris yielded more lipid than the intact form (33.6% compared to 10.0%), despite initially dissolving less into the IL liquor. This implies that the process of cell wall breaking does help downstream lipid extraction, allowing the IL to penetrate the cells and disintegrate lipids better. The pros and cons of the cell wall breaking pretreatment are discussed in the last section.
For the other ILs, the majority of the dissolved lipids stayed in the liquor, possibly due to the lipids still being bound within the lipid droplets, or bound to proteins, unextractable using hexane. Hexane was able to extract 2.5%, 4.5% and 7.2 wt% (intact C. vulgaris, broken C. vulgaris and S. platensis) of the total lipids out of the [Emim][OAc] liquors. These values are lower than the reported values for [Emim][OAc] pretreatment with C. vulgaris using other methods.24,25,28 It is worth noting that the extraction time was short (vigorous shaking followed by immediate centrifugation to separate the hexane layer), unlike other methods in which the IL/biomass mixtures were stirred with hexane for hours.25 Intact C. vulgaris proved difficult for all but [Ch][ARG], extracting yield for [Emim][OAc] and the other three amino acid ILs were all less than 5%. Significant yields were noted for [Ch][LYS]/S. platensis (27.1%) and [Ch][PHE]/broken C. vulgaris (12.0% yield).
One possible explanation for the effectiveness of [Ch][ARG] is the structure of the anion, as compared to the other IL anions. Studies have found that in applications involving ILs, the anion plays an important role in dissolving substrates, much more so than the cation.22 In lignocellulosic biomass pretreatment, the ILs that worked well all contained good H-bond acceptor anions e.g. Cl− or OAc−,22,40,42 that allow the ILs to bind to –OH groups that are present in the cellulose and proteins in the biomass. All amino acid anions have at least 3 hydrogen bonding sites, one from the amino NH2 and two from the carboxylate COO−. In this study, the argininate anion has three extra nitrogens on its branch, allowing a total of 6 hydrogen bonding sites to bind to the biomass. [Ch][LYS] also has one extra nitrogen on the anion, which might explain its better performance than [Ch][GLY]. [Ch][PHE] does not have any extra hydrogen bonding sites, but does have an aromatic phenyl ring, which suggests that π–π stacking might be playing a role in the interaction between [Ch][PHE] and the C. vulgaris cells.
C. vulgaris contained 296.4 mg carbohydrate per g of biomass (intact form), 133.4 mg g−1 (broken form) while S. platensis contained only 85.2 mg g−1 (Table 4). S. platensis is consumed as a dietary fibre supplement, credited to its fibrous and low calorie nature, in line with its lows lipid (102.7 mg g−1) and low sugar compositions. Pretreatment of C. vulgaris (both samples) produced solids richer in carbohydrates. Most exceptional was [Ch][ARG] which produced 800.7 mg g−1carbohydrate rich solid (equivalent to 82% starting sugars when solid recovery of 30.4% is taken into account). Combined with the lipid value, it is evident that this IL had carried out a highly precise fractionation of the biomass, for it has selectively dissolved the lipids of the biomass to produce a sugar rich solid. The effect was much more pronounced for the intact form of C. vulgaris than the broken form (362.8 mg g−1 sugar per dry solid residue), likely due to the carbohydrate storage (mostly as starch) in the broken cells being partially destroyed in the process of cell-wall breaking, consequently allowing free sugars to dissolve into the aqueous solution of the IL liquors. Other ILs produced solids less rich in carbohydrates than [Ch][ARG], but still retained 85–103% starting sugars from the intact C. vulgaris, and 52–79% from the broken C. vulgaris. It is clear that more sugar was lost from the broken C. vulgaris than the intact form. By contrast, in the case of S. platensis, only pretreatment with [Emim][OAc] preserves the sugar in the solid (94% sugar retained), choline-amino acid pretreatment resulted in a large loss of sugars. S. platensis has a softer cell wall than C. vulgaris, which must have allowed the sugars to dissolve into the aqueous solution of the choline amino acid ILs.
Carbohydrate (D-glucose + D-galactose) | |||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Chlorella vulgaris (Algomed, intact) | Chlorella vulgaris (Synergy, cell broken) | Spirulina platensis | |||||||||||||||
Raw | [Emim][OAc] | [Ch][ARG] | [Ch][GLY] | [Ch][LYS] | [Ch][PHE] | Raw | [Emim][OAc] | [Ch][ARG] | [Ch][GLY] | [Ch][LYS] | [Ch][PHE] | Raw | [Emim][OAc] | [Ch][ARG] | [Ch][GLY] | [Ch][LYS] | |
mg g−1 dry solid | 296.4 | 372.8 | 800.7 | 378.0 | 475.5 | 455.7 | 133.4 | 124.5 | 362.8 | 144.4 | 141.6 | 148.4 | 85.2 | 97.2 | 99.0 | 79.5 | 42.5 |
mg g−1 original biomass | 296.4 | 251.2 (85%) | 243.4 (82%) | 281.6 (95%) | 283.4 (96%) | 304.7 (103%) | 133.4 | 70.0 (52%) | 104.0 (78%) | 96.3 (72%) | 104.9 (79%) | 98.0 (73%) | 85.2 | 80.0 (94%) | 23.8 (28%) | 41.2 (48%) | 16.5 (19%) |
Enzyme mg g−1 original biomass | 97.0 (33%) | 133.5 (45%) | 210.0 (71%) | 194.0 (65%) | 173.7 (59%) | 162.3 (55%) | 28.8 (22%) | 13.5 (10%) | 31.3 (23%) | 29.5 (22%) | 24.2 (18%) | 28.2 (21%) | 19.8 (23%) | 11.9 (14%) | 22.2 (26%) | 15.3 (18%) | 13.9 (16%) |
The bioethanol production potential of raw biomass and their pretreated counterparts was estimated using enzymatic hydrolysis. A concoction of α-amylase, α-amyloglucosidase and cellulase was used to digest the biomass (as carbohydrates are present in both cellulose and starch). The amount ofD-glucose and D-galactose released per g of the original biomass (i.e. loss of solid during the pretreatment step was taken into account) is reported in Table 4 and Fig. 5. Unlike lignocellulosic biomass, which usually has a very high resistance to enzymes, the raw algal biomass did produce sugar upon enzyme hydrolysis, albeit not completely. Pretreatment was beneficial to the intact form of C. vulgaris, for all the IL pretreatment helped produce more sugar. [Ch][ARG] and [Ch][GLY] were exceptional and helped produce more than double the amount of sugars (released 71% and 65% of total sugars) as compared to the untreated sample (33%). On the other hand, for the broken form of C. vulgaris and S. platensis, there was not much improvement. This could be explained by the loss of sugar to the aqueous solution of the ILs. However, it is not clear why [Emim][OAc] pretreated S. platensis showed such a low activity towards enzyme hydrolysis, despite being richer in carbohydrate. [Emim][OAc] might have carried out inadequate fractionation, in which only the outer parts of the algae were removed while the core cell structure was still intact and resistant to enzymes; or there might have been strong attachment of the [Emim][OAc] molecules to the algal biomass which consequently poisoned and diminished the enzyme activity.
The question as to whether physical pretreatment is required remains open. As [Ch][ARG] produced the best results both in terms of lipids and sugar yields, it will be used to discuss the pros and cons of physical pretreatment. Cell disruption techniques for C. vulgaris (e.g. using pressure, ultrasonication, ozonation46 or enzymes47) certainly demand energy. Intact C. vulgaris fractionated better than broken ones, for 5.8% of total lipids and 82% of total sugars remained in the solid, cf. 12.5% and 78% for broken C. vulgaris. Enzymatic hydrolysis results were also better for the pretreated intact C. vulgaris (71% of total sugars was released compared to only 23% from the broken cells). However, the dissolved lipids of intact C. vulgaris were not easily recovered using hexane (only 10.0% was extracted, compared to 33.6% from the broken cells). Nevertheless, optimization of the extracting technique (e.g. longer immersion time with hexane, or addition of methanol as an anti-solvent instead of acid) might help improve the lipid yield. Taking everything into account, our opinion is that using intact cells and skipping any physical treatment offers a more economical solution. Optimization of the extraction step has to be investigated in future work.
This journal is © The Royal Society of Chemistry 2018 |