Selection of conditions for microwave hydrotropic extraction
We analyzed the effect of various combinations of conditions of microwave hydrotropic treatment, i.e. NaCS concentration (0, 10, 20% v/v), exposure time (10, 20, 30 min) and pressure (39, 78, 117 PSI) on the extraction of biomass components, changes in biomass composition (cellulose, hemicellulose and lignin content) and the amount of glucose released from pre-processed biomass as after hydrolysis with cellulases. The experiments showed that effective extraction of components of maize stillage biomass was possible even when only water was used as the solvent (0% v/v NaCS). This confirms the effectiveness of microwave treatment in the extraction processes. Lignocellulosic biomass extractives increased with exposure time and increasing pressure (Fig. 1). Higher pressure during the extraction was associated with higher temperature, which promoted dissolution of solid components. The extraction of biomass components also increased with increasing NaCS concentration, as a result of a decrease in the surface tension of the solution used. The highest biomass extractives, 67.00 ± 1.68%, were observed for the highest NaCS concentration (20% v/v), 30 min exposure time and 117 PSI (Fig. 1). Biomass extractives in this extraction variant were more than 12% higher compared to 10% v/v NaCS extraction and more than 20% higher than with water-only extraction, with the remaining process parameters unchanged (Fig. 1). Against the background of the current state of knowledge, the results showing effects of the combined application of microwaves and hydrotropes in plant biomass extraction are an element of scientific novelty. The positive influence of high temperature and elevated hydrotrope concentration on the extraction process was reported in previous studies. Increased temperature disrupts bonds in lignocellulosic biomass. This effect, in combination with the amphiphilic structure of hydrotropes and their ability to increase the solubility of organic compounds in aqueous solutions, increases the extraction [26]. In our earlier study, we also used the maize stillage biomass as a source of lignocellulose, but the maximum extractives were only 25.56 ± 1.25% at 131ºC (40.15 PSI) with 20% v/v NaCS [19]. The maximum extraction of biomass components from rice straw using 20% v/v NaCS at 131ºC was 31.07 ± 2.47% [21]. In the present study a much higher level of extraction of biomass components using NaCS was achieved due to the combined use of microwave radiation. The usefulness of microwaves in the extraction of biomass components was also confirmed for the NaOH solution. After vine shoots processing with microwave radiation and 3.1% w/v NaOH at 125ºC, biomass extractives increased to 54% [27]. Other authors also confirmed the dependence of extraction on the type and concentration of the solution used for pretreatment. In the microwave assisted treatment of Dragon Fruit, biomass extractives increased from ca. 4 to 11% and from ca. 7 to over 12% with increasing concentration of sulfuric acid (from 0.01 to 0.1 N) and sodium hydroxide (from 0.01 to 0.1 N), respectively [14]. A high level of extraction of vine shoot biomass components (ca. 53%) was also observed when microwave radiation was used along with NaOH solution under optimized process conditions, i.e. temperature (150ºC), time (30 min) and NaOH concentration (3.1% w/v) [28].
The microwave pretreatment with the use of a hydrotrope carried out in various process conditions caused changes in the composition of maize stillage biomass (Fig. 2). The use of water as an extraction solution during microwave treatment resulted in a partial extraction of organic matter from biomass. Water as the solvent did not have a major impact on the changes in cellulose concentration in biomass (average value around 37%), but it did lower hemicellulose levels and caused an increase in lignin concentration as extraction progressed, which was due to the non-polar structure of lignins (Fig. 2). The increase in lignin concentration was associated with the research methodology and resulted from the use of a constant amount of biomass after microwave extraction to analyze the biomass composition. The extraction with 10% v/v NaCS resulted in changes in the biomass content. The concentration of cellulose increased and the content of hemicellulose and lignins decreased as extraction progressed and pressure increased (Fig. 2). After 30 minutes of extraction with 20% v/v NaCS at 117 PSI this effect was enhanced: the concentration of cellulose, hemicellulose and lignins in biomass was 56.70 ± 0.26%, 1.55 ± 0.99%, 14.97 ± 0.32%, respectively (Fig. 2). The maximum reduction of lignin concentration in these conditions (20% v/v NaCS) was about 44% compared to the process using only water, with the remaining process parameters unchanged. The results leave no doubt as to the suitability of microwave radiation combined with the use of NaCS for maize stillage biomass delignification. The delignification effect is visible despite the high biomass extractives (about 67%). Because a constant amount of biomass is used to determine the components of lignocellulose, the effects of the lignin extraction process are somewhat covered up. To our knowledge, no reports describing microwave delignification using hydrotropes are available in the literature. However, many studies have confirmed that hydrotropes are effective in the extraction of lignins from plant biomass. Sodium xylene sulfonate (NaXS) used in the delignification process reduced lignin concentration by ca. 28% in eucalyptus biomass, by ca. 80% in sugar cane bagasse, by ca. 12% in wheat straw, and by ca. 47% in mixed birch and beech chips. Note that achieving this level of delignification required 30% NaXS applied at 110ºC (20 PSI) in sugar cane bagasse treatment or > 150ºC (> 68 PSI) in the treatment of other materials (wheat straw, a mixture of birch and beech chips, eucalyptus biomass); the exposure time was from1 to 8 hours [9, 20, 29, 30]. Studies demonstrated that NaCS could remove ca. 7 to 18% of lignins from distillery stillage biomass, ca. 30% from cotton stalks and ca. 52% from rice straw. Such reduction of lignin concentration was achieved for 20% v/v NaCS, at 131ºC in the extraction lasting at least 1 hour [19, 21, 31]. As a result of the combined use of microwave radiation with NaCS, as much as 44% reduction in lignin concentration was achieved in a very short time (at most 30 min); none of the previous studies reported such a large reduction in such a short time. The usefulness of microwaves in the delignification process was also confirmed by other studies. For pretreatment in combination with microwave radiation, sulfuric acid, ionic liquids or basic salts with phosphoric acid are most often used. Microwaves together with 0.5% v/v sulfuric acid reduced the concentration of lignins in aloe biomass by up to 66% [22]. The combined action of ionic liquids together with microwave radiation at 130ºC resulted in a nearly 80% reduction in lignin concentration in Miscanthus biomass and birch wood [24]. Pretreatment with microwaves and basic salts in phosphoric acid reduced the lignin concentration in rice straw by only 12 to 20% [16]. Similarly to our study, it was found that as a result of extraction of organic substances from plant biomass, the concentration of cellulose increased.
Biomass samples after hydrotropic microwave delignification were also subjected to enzymatic hydrolysis using a cellulolytic enzyme. The purpose of this research stage was to determine the effect of various process conditions of microwave hydrotropic delignification on glucose concentration after enzymatic hydrolysis of cellulose. Studies confirmed a high efficiency of hydrolysis of cellulose in maize stillage biomass after microwave hydrotropic delignification. The highest glucose concentration in the hydrolyzate was over 450 mg/g DW. This concentration was achieved after microwave treatment with NaCS under conditions providing the highest biomass reduction and the highest cellulose concentration, i.e. at 20% v/ v NaCS, 117 PSI and 30 min exposure time (Fig. 3). Obtaining such a high glucose concentration per gram of biomass after pretreatment was not possible in our previous studies on the same raw material, which was subjected to barothermal treatment combined with diluted sulfuric acid [32]. In that experiment, a maximum of about 225 mg glucose per gram of biomass was achieved. However, the use of dilute sulfuric acid together with microwave radiation resulted in ca. 270 mg of glucose per gram of biomass after cellulose hydrolysis [11]. The susceptibility of biomass after hydrotropic treatment to enzymatic hydrolysis using cellulases was also demonstrated in studies on eucalyptus biomass. In eucalyptus biomass pretreated with NaXS, the conversion of cellulose to glucose was as high as about 80% [9].
Optimization of enzymatic hydrolysis of cellulose in biomass after microwave hydrotropic delignification
The next stage of the study after choosing the parameters of microwave hydrotropic delignification was the optimization of conditions for cellulose enzymatic hydrolysis. To this end, maize stillage biomass previously subjected to microwave hydrotropic treatment with 20% v/v NaCS at 117 PSI for 30 minutes was hydrolyzed using a cellulase preparation in acetate buffer pH 5.5. The hydrolysis process was analyzed for three biomass concentrations (4, 8, 16% w/v) and three enzyme dose levels (1, 2, 4 FPU/g DW biomass). All experiments were run for 72 hours. The highest cellulose hydrolysis yield, about 80%, was obtained for 4% w/v biomass concentration at the highest enzyme dose (4 FPU/g biomass) after 48 hours of the process (Fig. 4). Importantly, there were no statistically significant differences in cellulose hydrolysis yield between different biomass concentrations (4, 8, 16% w / v). at a given hour using a given dose of enzyme. A clear increase in cellulose hydrolysis yield in subsequent hours of the process was observed only for enzyme doses of 1 and 2 FPU/g biomass. The yield difference between the 24th and 72nd hour of hydrolysis at a dose of 1 FPU/g biomass ranged from about 30% for 4% w/v biomass concentration to about 20% for 16% w/v (Fig. 4). For the enzyme dose increased to 2 FPU/g biomass, the yield difference between 24th and 72nd hour of hydrolysis was around 20% regardless of the biomass concentration used. At the highest enzyme dose, 4 FPU/g biomass, the yield of cellulose hydrolysis after 72 hours was only 8% higher than after 24 hours of the process, regardless of the biomass concentration in the medium (Fig. 4). Conditions that ensured high glucose level after 24 hours of hydrolysis, i.e. 16% w/v biomass concentration and enzyme dose of 4 FPU/g biomass, were selected for the next stage of the study. In our previous studies on the same raw material after baro-thermal treatment in a dilute sulfuric acid environment, the maximum yield of cellulose hydrolysis was 70% (it was lower by ca. 10% compared to this work), but only after 72 hours of the process [32]. In studies using corn stover, the highest cellulose hydrolysis yields were reported only at the highest enzyme concentration (40 mg protein per 1 g of glucan) and the highest biomass concentration (20% w/v). At lower enzyme concentrations, a significant decrease in hydrolysis yield was observed, even by 35% [33]. Also authors using rice straw as lignocellulosic raw material pointed out that obtaining the highest hydrolysis yield required precise optimization of process parameters. Properly selected enzyme and biomass concentrations resulted in up to 86% cellulose hydrolysis yield [34].
Evaluation of the suitability of hydrolyzate from maize stillage after microwave hydrotropic treatment and enzymatic hydrolysis for the production of cellulosic ethanol
At the last stage of the study, maize stillage biomass after microwave hydrotropic treatment and enzymatic hydrolysis was used to produce second generation ethanol. The hydrotropic microwave treatment with NaCS proved to be a very effective way of preparing maize stillage biomass for fermentation. It is worth noting that the proposed simplified pretreatment, involving only NaCS and microwaves (without additional acid treatment) allows to obtain a fermentation medium containing up to 80 g of glucose per liter, without condensing. This result was achieved after enzymatic hydrolysis of lignocellulose components present in 160 g/L of biomass (Fig. 5). Eliminating the need to condense the substrate to obtain a high concentration of fermentable sugars is an important achievement of the proposed method. It suggests that microwave hydrotrope can improve the economics of cellulosic ethanol production. In the simplest experimental variant (MHF 1) only cellulolytic enzymes from Cellic® CTec2 preparation were used to hydrolyze cellulose after pretreatment of biomass. In order to create optimal conditions for these biocatalysts, acetate buffer pH 5.5 was used as the solvent. In other experimental variants (MHF2 and 3), in addition to Cellic® CTec2, hemicellulose degrading enzymes were used to increase the amount fermentable sugars, i.e. glucose and galactose. In these variants, acetate buffer pH 5.5 (MHF 2) or water pH 5.5 (MHF 3) was used as the solvent (Table 1). The lowest initial concentrations of glucose, ca. 69 g/L, as well as galactose and xylose, ca. 3.5 g/L, were observed in in the MHF 1 variant, because in this experiment only cellulolytic enzyme was applied (Table 1, Fig. 5, 6). When additional hemicellulose-degrading enzymes were used, a higher glucose concentration (ca. 79 g/L) was obtained, irrespective of the solvent used (buffer or water); galactose and xylose level also increased to ca. 6 g/L (Fig. 5, 6). Arabinose was not found in any of the fermentation media. It is worth emphasizing that fermentation media obtained after microwave hydrotropic treatment of maize stillage biomass did not contain 5-HMF, furfural and lignin degradation products such as syringaldehyde, trans-ferulic acid, vanillin, 4-hydroxybenzoic acid (Table 2). The initial phase of fermentation in such media was not impeded, as observed in media with inhibitors. The undisturbed course of the initial fermentation phase resulted in complete bioconversion of glucose to ethanol after 48 hours of the process, in each experiment (Fig. 5). In experiments with Viscozyme® L (MHF 2 and 3), between 24 and 48 h of fermentation, only galactose was fermented, which led to a decrease in the sum of this sugar and xylose by ca. 0.5 g/L (Fig. 6). Data collected during fermentation experiments suggest that is possible to achieve relatively high ethanol concentrations in lignocellulosic media, e.g. 35.54 ± 1.42 g/L for MHF 1 and 3 ok. 41.5 g/L for MHF 2 (Fig. 5). Statistical analysis showed no significant differences between the initial glucose, galactose and xylose concentrations and the final ethanol concentration in MHF variants 2 and 3. The absence of fermentation inhibitors in the media combined with the high initial concentration of fermenting sugars resulted in a high concentration of ethanol and high fermentation yield. After 72 hours of the process, the ethanol yield in relation to the theoretical one was about 95%, in all variants (Table 3). High yeast activity also exerted an effect on glycerol concentration in fermentation media (Fig. 6). This work demonstrated that maize stillage biomass after initial microwave hydrotropic treatment and enzymatic hydrolysis can be used to produce second generation ethanol. It is worth noting that achieving a high attenuation level did not require any supplementation with mineral substances or additional source of nitrogen, which otherwise would also affect the profitability of the process.
Table 1
Charakterystyka podłoży fermentacyjnych przygotowanych w trakcie badań.
Research variant | Solvent | pH | Enzyme preparation |
MHF 1 | 0.05 M acetate buffer | 5.5 | Cellic® CTec2 |
MHF 2 | 0.05 M acetate buffer | 5.5 | Cellic® CTec2 Viscozyme® L |
MHF 3 | water | 5.5 | Cellic® CTec2 Viscozyme® L |
Table 2
Początkowe stężenie 5-HMF, furfuralu oraz produktów degradacji lignin (syringaldehyde, trans-ferulic acid, vanillin, 4-hydroxybenzoic acid) w podłożach fermentacyjnych.
Research variant | Initial concentration of phenolic compounds (mg/L) in the fermentation medium | Initial concentration of 5-HMF (mg/L) in the fermentation medium | Initial concentration of furfural (mg/L) in the fermentation medium |
syringaldehyde | trans-ferulic acid | vanillin | 4-hydroxybenzoic acid |
MHF 1 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
MHF 2 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
MHF 3 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Table 3
Wydajność fermentacji alkoholowej względem wydajności teoretycznej po 72 godzinach procesu.
Research variant | Yield of alcoholic fermentation [% of theoretical yield of glucose, galactose and xylose] after 72 hours |
MHF 1 | 95.75a ± 0.86 |
MHF 2 | 94.69a ± 0.61 |
MHF 3 | 95.33a ± 0.57 |
The mean values given in columns with different letter index are significantly different (α < 0.05). |
A review of the literature indicates the innovative nature of the proposed technological solution to use microwave radiation along with hydrotropes to process lignocellulosic biomass in ethanol production. Only a few studies attempted to use plant biomass after hydrotropic treatment at elevated temperature and pressure in the production of cellulosic ethanol. However, no previous study has achieved such a high concentration of fermentable sugars in the culture medium as in the present work. For example, the fermentation medium prepared from cotton stalks after hydrotropic treatment contained only 5.15 g of reducing sugars per liter [31]. The use of NaCS and NaXS in the processing of rice straw delivered 19.74 g and 10.22 g of glucose per liter of fermentation medium, respectively [21]. In previous studies, we used NaCS to pretreat maize stillage biomass. The process was carried out at 131ºC for 1 hour. The final glucose concentration in the fermentation medium was about 63 g/L, but only after condensing [19]. Other authors reported that rice straw after hydrotropic treatment can be a source of cellulose susceptible to enzymatic hydrolysis and could provide an ethanol yield of 73 or 78% relative to the theoretical yield depending on the hydrotrope used. They pointed out that effective removal of hydrotropes from biomass is important, because residues of these compounds at higher concentrations might adversely affect the yeast metabolism [21]. Hydrotropic treatment can also be useful in the preparation of wheat straw in the butanol biosynthesis process: the use of NaXS provided ca. 23 g/L glucose and, after conversion, over 9 g of butanol per liter [30].