3.1 Fractionation yield
The fractionation yields of 1:50 and 1:100 ratios were around 60%, with a slight tendency to reduce as a function of lower water content, as following: 63.70% (1:100 ratio), 59.40% (1:50) and 52.40% (1:35). These yields corroborate with previous studies [18, 21]. We observe a smaller fractionation yield was observed for the ratio 1:35, which was motivated by less water amount and more difficulty in the bio-oil demulsification. Similar effect of the amount of bio-oil added to water was observed by Matos et al. They defined 10 g per liter as the optimal condition for an efficient separation in cold-water [9]. Also, water content less than 85% are inefficient to fractionate high-viscosity bio-oil under lower temperatures [12]. Specifically, our bio-oil has high viscosity (Table 1), which partially corroborates with these statements. Then, the water-soluble fraction produced considering the ratio of 1:35 was discarded for subsequent analysis.
There is no consensus on a standard protocol for bio-oil fractionation and its yield. For instance, Zhang and Wu [12] found a yield of 35.56% for the water-soluble fraction after bio-oil fractionation considering the oil-to-water ratio of 1:20 and temperature of 20 ºC. Similarly, there is no accordance for the most appropriated agitation speed. An efficient fractionation requires vigorous agitation since the physical contact between the particles can stimulate the agglomeration of the particles and their surface charge reduction [22]. We understand the high-shear agitation by Ultra-Turrax, low temperature, and adequate oil-to-water ratios used in our study contributed positively to better depolymerization of the chemical compounds of the bio-oil, making part of them soluble in water. Matos and co-authors have observed recently non-shear agitation (10 g L− 1 and 2k rpm) prevents the bio-oil separation. They pointed out the necessity to break the drops of bio-oil for an efficient separation [9].
Table 2
Physicochemical properties of water-soluble fractions produced with oil-to-water ratios of 1:50 and 1:100.
Property
|
1:50
|
1:100
|
Density (g/cm³)
|
0.9988 (0.0016)
|
0.9990 (0.0011)
|
Concentration (g/L)
|
8.69 (0.12)
|
4.20 (0.16)
|
Values between parentheses correspond to standard deviation. |
As expected, the concentration of solids of the water-soluble fractions increases with the decreasing water content used in the fractionation. In turn, the density at 20 ºC remained similar to pure distilled water for all conditions of fractionation, reaching 0.99 g/cm³.
3.2 Chemical composition by GC-MS
GC-MS analysis was performed to investigate the effect of fractionation conditions on the nature and type of chemical compounds presented in the water-soluble fraction. We only considered peaks with a high degree of probability (≥ 80%). We identified chemical compounds considering the water-soluble fractions for the two oil-water ratios, and they were grouped according to the highest priority organic function (descending order: carboxylic acids, esters, aldehydes, ketones, alcohols, phenols, ethers and alkanes).
Chemical distribution depended on the extraction methods. We have observed specific differences due to the chemical affinity of the compounds with the organic solvents.
Any chemical compound identified in the GC-MS was sensible for all extraction methods. But some chemical compounds were identified through more than one extraction method, for example, vanillin (r.t. 15.173). phenols, such as catechol, eugenol and 3,5-Dimethoxy-4-hydroxytoluene are also substantially presented in the water-soluble fractions. They are generated from the thermal degradation and cleavage of lignin units commonly presented in the biomass, which are known to resist high temperatures during the fast-pyrolysis process [23, 24].
Although demulsification separates insoluble- and soluble-water compounds, part of these phenols remain in the water-soluble fraction since they are moderately soluble in this medium. This corroborates with the findings of Ren et al. [18]. The most propitious extractions methods to identify phenols in the water-soluble fraction was ethyl acetate, dichloromethane, and drying in a SpeedVac vacuum concentrator. The tendency to remain in the water-soluble fractions highlight this product as an alternative for antioxidant applications. Similarly, ketones from the thermal degradation of cellulose and hemicelluloses [25] remain substantially in the water-soluble fraction due to their polar nature.
On the contrary, anhydrosugars are only identified in the extraction method of drying in SpeedVac vacuum concentrator. These chemical compounds are related to the thermal degradation of cellulose and its dehydration due to high temperatures – near to 600 ºC - applied in the fast-pyrolysis process. They can represent around 39% of the total content of the bio-oil [26, 27]. The anhydrosugars identified in the water-soluble fractions were 4-O-β-D-galactopyranosyl- α-D-glucopyranose (r.t. 17.607), levoglucosan (r.t. 16.553), 1,4:3,6-Dianhydro-α-D-glucopyranose (r.t. 12.510) and 2,3-anhydro-D-mannosan (12.740), with a notable presence of levoglucosan.
Oxygenated compounds belonging to the chemical class of aldehydes, such as furfural (r.t. 4.985) - originated from dehydration of carbohydrates - and phenolic compounds like catechol (r.t. 12.116) and vanillin (r.t. 15.173) – originated from the lignin degradation – were also identified in the water-soluble fractions, since they are commonly found in the raw fast-pyrolysis bio-oil [2, 28–30]. Also, the remarkable presence of catechol in the water-soluble fractions denotes its solubility in water [31].
In summary, we observed the remarkable permanence of polar chemical compounds after fractionation in cold water, the chemical composition of the aqueous fractions of the bio-oil are often similar [33]. Solubilization of polar compounds in the water-soluble fraction occurs due to their low molecular weight. Part of these chemical compounds attracts the interest of industries, such as catechol, levoglucosan, vanillin, and 5 - hydroxymethylfurfural. For instance, levoglucosan originated from renewable raw materials has been attracting attention because of its complex synthesis by synthetic routes, and as an alternative to produce monomeric sugars, mainly D-glucose [32]. Furthermore, levoglucosan presents useful characteristics demanded by the chemical industry for the production of plastics, surfactants, resins, and substitution of sorbitol, such as hydroxyl groups at the C-1 anomeric center position, the primary hydroxyl group at C-6, and chirality [33, 34]. We also highlight the presence of catechol (1,2-dihydroxybenzene), one of the most important phenolic compounds of the water-soluble fractions [35] originated from the lignin thermal degradation. Containing a benzenic ring, catechol has remarkable antioxidant activity due to its ability to chelate metallic ions, and high antimicrobial potential [36].
With the method of drying in a SpeedVac vacuum concentrator we observed the presence of high value-added chemicals in this fraction, such as vanillin used as a flavor, catechol with known antioxidant potential, and levoglucosan commonly used to produce antibiotics and antiparasitic agents [37–39]. Then, we select this fraction generated by drying in a SpeedVac vacuum concentrator to evaluate its physicochemical stability (Fig. 1).
3.3 Physicochemical stability of the water-soluble fractions
The low thermal stability is one of the most relevant bio-oil limitations [40]. Considering this well-known instability of the crude bio-oil used as raw material in this study, and presupposing a similar behavior of the water-soluble fractions, as previously described by Meng et al. [41], we decided to investigate by GC-MS its stability and chemical reactivity during nine days of storage at 40 ºC (Fig. 2 ). The main results denote most compounds showed chemical stability and a slight instability of some compounds present in the water-soluble fractions. The substantial presence of carboxylic groups (C = O) in the water-soluble fractions can influence this storage stability, since they are highly reactive, impacting directly on the intrinsic chemical reactions during the accelerated aging [42].
Compounds from the group of anhydrosugars, 1,4:3,6-Dianhydro-.alpha.-d-glucopyranose (1,4:3,6 – D alpha d-g), 4-O-.beta.-D-galactopyranosyl- alpha.-D-Glucopyranose, (4-O beta D-g alpha D-G) and beta.-D- Glucopyranose, 1,6-anhydro- (levoglucosan), exhibited moderate instability of the water-soluble fractions, represented mainly by levoglucosan, with little variation in peak area during storage, especially at the 1:50 oil-water ratio. Levoglucosan can participate in polymerization reactions or can be hydrolyzed to form glucose [44]. Cathecol, methoxyeugenol and hydroxyeugenol, compounds pertains to the phenolic group, showed statistical stability. These compounds have great antioxidant activity and antimicrobial potential [37].
This suggests water-soluble fractions as a renewable alternative in a biorefinery platform for levoglucosan separation and further hydrolysis to glucose monomers, or even sugar recovery, both notable feedstocks for fine chemical applications. We highlight the low losses in the relative area after nine days of storage, which partially guarantees the chemical stability of the anhydro-sugars in the water-soluble fractions. In other words, aging did not result in substantial degradation of anhydrous sugars such as levoglucosan. In contrast, the most part of phenolic and aldehyde compounds remain statistically stable during aging for nine days at 40°C, with no changes in the relative peak area.
Based on these preliminary results of 9 days of storage stability, a new accelerated aging test for 43 days was carried out in the 1:50 water-soluble fraction due to the abundant presence of high added value compounds previously evidenced, with special attention anhydrosugars and phenolic compounds (Table S1). We followed similar conditions of accelerated aging at 40 ºC with evaluation in three periods, 1, 30 and 43 days for selected compounds with analysis in GC-MS in Selected Ion Monitoring (SIM) mode (Fig. 3).
Comparing with nine days, we observed similar behavior of the water-soluble fraction 1:50 after 43 days of storage, the relative area of the majority of phenols and aldehydes remained statistically stable (Fig. 3-A). The stability of most of the groups of compounds present in the water-soluble during storage may be related to its lower reactivity and lower concentration compared to crude bio-oil. The chemical interactions and reactions between fractions of the bio-oil are the most critical reasons for its aging. Organic acids presented in the water-soluble fractions can promote the condensation of lignin fraction and degradation of sugars. Furthermore, aldehydes. Furthermore, compounds that were not significantly stable not degrade completely (Fig. 3 -B) can interact with pyrolytic lignin (water-insoluble fraction), resulting in its reticulation [42]. Thus, separation of insoluble- and water-soluble fractions in a proper time can contribute to mitigating these interactions and reactions, providing materials with desirable characteristics.
In summary, we pointed out the possibility to storage water-soluble fractions without substantial losses for separation and purification of high value-added compounds in a biorefinery strategy, especially anhydrosugars, phenols and aldehydes (Fig. 3). Considering both positive and negative aspects found in the accelerated aging and the operational conditions for fractionation used in this study, we suggest the storage of water-soluble fraction of fast-pyrolysis bio-oil for no more than 43 days at risk of reducing yield of the high value-added chemical compounds. This alternative of recovery of chemical compounds from water-soluble fractions can contribute to reduce the use of fossil sources, and the generation of residues from biomass-based sector [43].
3.4 Thermal behavior
Three thermal mass loss events for both oil-water ratios with similar thermal degradation up to 800°C (Fig. 5). The first event occurs up to 100°C and refers to the evaporation of wastewater molecules and low molecular weight volatile organic compounds, both are commonly presented in the water-soluble fractions. The mass loss in this first event was up to 5%.
The second event refers to thermal degradation of common chemical compounds generated during the fast pyrolysis of the biomass, especially from the cellulose and lignin degradation. This event starts at 180 ºC, which was the onset temperature in all thermograms, and it remains with mass loss up to 600 ºC. In this event, we observed substantial mass loss of 70% due to the chemical composition of the water-soluble fractions described in Fig. 3. For instance, levoglucosan is the dominant chemical compound in the water-soluble fractions and degrades around 500 ºC [44].
The third event occurs between 650 ºC and 800 ºC with less mass loss. This event refers to the formation of charcoal generated from the degradation of more thermally stable compounds. The residual mass loss of the two water-soluble fractions was close to 25%. This suggests coke formation due to the polymerization of phenols and alcohols during pyrolysis [47].
3.5 Phenolic total content and antioxidant activity
Table 3 shows the antioxidant activity and the total phenolic content of the water-soluble fractions. The DPPH free radical scavenging capacity of the water-soluble fractions was expressed in terms of IC50 and EC50. We observed strong antioxidant activity of all 1:50 and 1:100 water-soluble fractions, with 1–2 mg to reduce the same amount of DPPH radical. The 1:100 oil to water ratio exhibited the strongest DPPH free radical scavenging ability.
Table 3
Total phenolic content and antioxidant activity evaluated with DPPH, ABTS and FRAP assays of water-soluble fractions.
Oil-to-water ratio
|
Antioxidant activity
|
Total phenolic content
(mg GAE/g)
|
DPPH
|
ABTS+
|
FRAP
|
IC50
(mg/L)
|
EC50
(mg/mg DPPH)
|
Eq. 1000 uM of Trolox (mg/L)
|
Eq. 1000 uM of FeSO4 (mg/L)
|
1:50
|
39.87
(4.88)
|
1.74
(0.21)
|
1382.72
(95.48)
|
1306.21
(9.56)
|
79.41
(2.49)
|
1:100
|
26.64
(1.81)
|
1.16
(0.08)
|
1415.46
(34.90)
|
1074.28
(11.83)
|
86.92
(1.55)
|
Compared to the products made with butylhydroxytoluene (BHT) and butylated hydroxyanisole (BHA), the water-soluble fractions exhibited relevant activity to scavenging hydroxyl free radical. BHT and BHA are synthetic oxidants with adverse effects widely applied in dermo-cosmetics and food industries. The BHT and BHA present EC50 of 0.038 mg/mL and 0.056 mg/mL, respectively [48], both comparatively lower than the EC50 found in water-soluble fractions.
The ABTS assay detected the high antioxidant activity of all water-soluble fractions, with the strongest capability in the fraction produced with oil-to-water ratio of 1:100. ABTS + method can efficiently describe the antioxidant activity of samples rich in polar and non-polar compounds [49]. We have confirmed this characteristic by the chemical composition of the water-soluble fractions, as previously illustrated in Fig. 6.
FRAP assay confirmed the potential application of water-soluble fractions as an antioxidant agent. However, the higher ferric reducing power was found in a water-soluble fraction 1:50, followed by a 1:100 fraction. This suggests lower water content for the bio-oil fractionation, and a higher concentration of solids can positively influence the reduction of ferric ions (Fe(III)). This result corroborates with the ferric reducing power found in the raw bio-oil, such as for slow-pyrolysis bio-oil found by Wei et al. [50].
Overall, we suggest this strong antioxidant activity for the abundant number of phenolic compounds previously observed in water-soluble fractions via GC-MS. We detected high that most of the chemical compounds that constitute WS are phenolic in the water-soluble fractions, 1:50 and 1:100. This behavior can be confirmed by the total phenolic content (Table 3). The water-soluble fractions had a total phenolic content of 79–87 mg/g gallic acid equivalent (GAE). Furthermore, we found a positive correlation with the water content used in the fractionation step, confirming the findings of Zhang and Wu [12]. This positive relationship between phenolic compounds and antioxidant activity was also observed by Lu et al. [51]. But they found no significant correlation of the antioxidant activity of rapid pyrolysis bio-oil with ketones, alcohols, aldehydes and esters.
The strong antioxidant activity of the water-soluble fractions can be attributed in part to molecules such as syringol (2,6-dimehoxiphenol) eugenol and catechol, present in the WS (Table S2). since they have a high content of hydrogen atoms they stabilize free radicals through the donation of hydroxyl groups [52, 53]. In summary, despite the high dilution, the water-soluble fractions of all two oil-water ratios exhibited strong and relevant antioxidant activity, especially due to the phenolic compounds found naturally in the material and compared to synthetic antioxidant agents.
3.6 Fungal inhibition
Disc diffusion agar experiments revealed growth inhibition of the fungi Trametes Versicolor and Gloeophyllum trabeum. The two water-soluble fractions exhibited high antifungal activity, mainly in the 1:50 fraction (Fig. 6).
The water-soluble fractions showed strong growth inhibition compared to the control, in which the latter needed only 10 and 14 days to completely remove the plaque for the fungi T. versicolor and G. trabeum, respectively. The water-soluble 1:100 fraction showed 44% inhibition for T. versicolor and 20% for G. trabeum after 26 days of exposure. In contrast, the 1:50 water-soluble fraction completely inhibited the growth of both fungi.
The total phenolic content partially supports the inhibition of the growth of both fungi by the water-soluble fractions. Phenolic compounds such as catechol and eugenol alter the composition of the fungus's plasma membrane [54]. Phenolic compounds have antioxidant activity and can chelate metallic ions, since the action of fungi occurs through oxidative attack and the breaking of hydroxyl radicals present in the chemical structure of wood [55–57]. Furfurilated wood, in turn, has good resistance to decay due to the presence of furfural and 5-hydroxymethifurfural in its composition [58, 59]. Furthermore, the presence of organic acids identified via GC-MS (Fig. 6C and Table S4) may contribute to fungal inhibition. Organic acids from cellulose degradation - such as propionic acid found in our water-soluble fractions - can block the energy production mechanism of fungi through acidification of the cytoplasm [60, 61], reducing wood decomposition [62, 63]. Thus, previous studies have reported the individual or combined effect of phenolics, organic acids and other chemicals such as fatty acids play an important role in protecting wood against fungi [64–66]. However, the total solids concentration (see Table 2) of the water-soluble fractions can lead to fungal inhibition in our study. Even the 1:50 fractions had 5–9% less phenolic content than the 1:100 fraction, their 2–3 times higher solids concentration just indicates the presence of chemicals such as organic and phenolic acids are not sufficient to provide a total inhibition of fungal growth.
3.7 Bacterial inhibition
The three water-soluble phases presented similar antibacterial activity against the gram-negative bacteria E. coli and gram-positive S. aureus (Table 4). The minimum inhibitory concentration (MIC) for the two water-soluble phases was the same for both bacteria of 2,10 and 2.17 g/L. This suggests the potential of simultaneous application of the water-soluble phases for both bacteria since they need the same concentration to inhibit equally the microbial growth.
Table 4
Minimum inhibitory concentration against E. coli and S. aureus bacteria.
Minimum inhibitory concentration (g/L)
|
S. aureus
|
E. coli
|
Antibiotic
|
Amoxicillin
|
Trimethoprim
|
0,12
|
0,62
|
1:50
|
2,17
|
2,17
|
1:100
|
2,10
|
2,10
|
This remarkable antibacterial activity of the water-soluble phases can be justified by the presence of phenolic compounds, especially those with hydrophobic nature. These compounds are capable to deposit in the cellular membrane of bacteria, causing leakage of intracellular content and subsequent death of the microorganism [69].
Furthermore, the antibacterial potential of the raw bio-oil - source of the water soluble phases - has been widely studied [69–71]. They report this activity to the furans, ketones, acids, and many carboxylic groups presented in the bio-oil. We highlight the presence of all these chemical compounds in the water-soluble phases, such previously identified by GC-MS. Nevertheless, the antimicrobial potential may not be attributed
only to a group of chemical compounds, but to synergy between them [72]. Overall, the three water-soluble phases generated by a simple bio-oil fractionation process presented relevant MIC against all tested bacteria, denoting their high potential for antimicrobial applications.