Coproduction of cellulosic ethanol, lignosulfonate, and phenolic acids from an integrated corn cob valorization scheme

doi:10.21203/rs.3.rs-3405666/v1

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Coproduction of cellulosic ethanol, lignosulfonate, and phenolic acids from an integrated corn cob valorization scheme

https://doi.org/10.21203/rs.3.rs-3405666/v1

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published 20 Feb, 2024

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The economic competitiveness of 2G-bioethanol technology should improve through the use of by-products, especially lignin. The present study developed an integrated scheme to produce 2G-bioethanol, lignosulfonate, and phenolic acids from corncobs. Pretreatment with sulfuric acid made it possible to reduce the enzymatic dosage to 5 FPU/g in the enzymatic hydrolysis of samples with a size of less than 20 mesh. The addition of polyethylene glycol 1500 (PEG 1500) boosted the sugar release, reaching 56.73 g/L glucose under conditions with 20% (w/v) solids (gradual addition). In the ethanol production, the semi-simultaneous saccharification and fermentation strategy achieved a maximum production of 31.64 g/L in the condition with 5 FPU/g, 2% (w/w) PEG 1500, and 20% (w/v) solids (gradual addition). FTIR confirmed the preparation of lignosulfonate from fermentation residue, and the surfactant showed good stabilization performance in oil/water systems. Hydrogen peroxide promoted high yields of p-coumaric acid (8045.3 mg/100 g) and ferulic acid (1429.4 mg/100 g) in the alkaline hydrolysis of fermentation residue. Based on the results, corncob is versatile and can be used to create a biorefinery with high economic potential.

Second generation

biofuels

enzymatic hydrolysis

surfactant

p-coumaric acid

The main sources for the industrial production of first-generation bioethanol are currently sugarcane and corn [1]. In 2022, annual production of first-generation bioethanol is estimated at 110 billion liters, with the United States being the main player in the market [2]. Without a doubt, first-generation technology has been necessary for energy security and has supported the reduction of carbon emissions in the transport sector [3]. However, the first-generation technology requires significant amounts of water, fertilizers, and arable land, creating a dilemma between two fundamental human needs: food and energy [4]. Given the current critical state of global water and food resources, it has become imperative to explore more sustainable approaches for producing bioethanol [5].

Corn (Zea mays) is one of the world's most widely cultivated cereal crops. Its production for human and animal consumption, as well as for ethanol, creates a significant amount of waste [4]. According to Potumarthi et al. [6], for every kilogram of dry corn grain produced approximately 0.3 kg of corn cob are collected. Between 2018 and 2019, global corncob generation was estimated at 3.1 × 10⁷ tons, representing high environmental stress if discarded inappropriately [7]. Luckily, corncob has been used for different purposes, including second-generation ethanol. Corncob has a high holocellulose content (~ 61%-74%) and low lignin content (16%-20%), being easily transformed into bioethanol via the biochemical route [8, 9]. In Gandam et al. [10], the chemical composition of corncob is highlighted for second-generation ethanol production when compared to other lignocellulosic biomasses.

One of the biggest challenges to converting lignocellulosic biomass into ethanol is the natural barrier of plant cell walls, which hinders the efficient hydrolysis of cellulose into fermentable sugars [11]. To overcome this obstacle, pretreatments are required, but this can add up to 20% to the total cost of the process [12]. Furthermore, producing second-generation ethanol requires more enzymes than first-generation ethanol. [13, 14]. The addition of amphiphilic polymers is an attractive path to reduce the consumption of cellulases. These additives were inserted into the production chain to reduce the non-productive adsorption of cellulases onto lignin and reduce deactivation at the air-liquid interface [7, 11, 15]. To make viable and to enhance the profits of cellulosic bioethanol plants from corncob, a combined refining technology that makes full use of cellulose, hemicellulose, and lignin (three main components of lignocellulosic material) to produce high-value co-products is also mandatory.

Based on techno-economic analyses, it has been determined that only 40% of the generated lignin is necessary to meet the energy demand of a regular biorefinery by combustion [4]. As the trend for biorefineries is on the rise, lignin production is expected to increase as well; therefore, it is crucial to optimize its usage. Lignin has been reported as a valuable material for multiple applications, but generally it must be previously functionalized. Lignosulfonates, also known as sulfonated lignin, are obtained via sulfite pulping techniques employing a sulfurous acid solution containing either sulfites or bisulfites [16]. This process results in the cleavage of linkages between lignin and carbohydrates, as well as the C–O bonds that connect the lignin units, followed by the replacement of the hydroxyl group into the lignin structure by the sulfonate group [17]. Lignosulfonates can act as animal feed binders, concrete additives, oil well additives, and dust control [18].

Due to their impressive qualities, phenolic acids derived from lignin, including ferulic and ρ-coumaric acid, have been reported for potential use in food, health, cosmetic, and pharmaceutical industries [19]. In the cosmetic area, the acids act against cutaneous damage and diseases such as skin cancer [20]. Ferulic acid, a natural antioxidant, has been found to neutralize free radicals and offers numerous benefits such as antioxidant, antimicrobial, anti-inflammatory, antidiabetic, and anti-carcinogenic properties [21]. p-Coumaric is also a plant-based antioxidant with antioxidant activity, antidiabetic, and anti-carcinogenic properties [22]. Furthermore, alkaline hydrolysis to obtain phenolic acids, which has been used for several years [23, 24], can be fully integrated into the second-generation bioethanol technology, using the accumulated lignin in solid waste as a precursor.

Therefore, the present study developed an integrated scheme to value corncobs. Acid pretreatment with dilute sulfuric acid was selected to improve enzymatic digestibility. Operating conditions were changed to maximize sugar and ethanol production. Discussions were raised about using polyethylene glycol (a polyether with hydroxyl groups at the ends) to mitigate enzyme consumption in an environment with a solid loading of up to 20% (w/v). Furthermore, the solid residue from corncob fermentation was converted into lignosulfonate and phenolic acids (p-coumaric acid and ferulic acid).

Figure 1 shows the integrated scheme developed to convert corncob into value-added products. The first step of the research focused on selecting the particle size and the enzyme dosage to achieve high digestibility and fermentability of the corncob. In the second step, the residue from saccharification and simultaneous fermentation was used to obtain lignosulfonate via sulfomethylation and phenolic acids via alkaline hydrolysis. The subsections below show in detail the valorization procedures.

2.1. Chemicals

Citric acid, sulfuric acid, sodium citrate, ethanol, yeast extract, glucose, sodium hydroxide, n-octanol, peptone, hydrogen peroxide, and toluene were purchased from Synth (São Paulo, Brazil). Polyethylene glycol with an average molecular mass of 1500 (PEG 1500) was also purchased from Synth (São Paulo, Brazil). Formaldehyde and sodium bisulfite were purchased from Vetec Química Fina (Rio de Janeiro, Brazil). The standards p-coumaric acid (crystalline) and trans-ferulic acid (99%) were purchased from Sigma-Aldrich (MO, United States). Soybean oil was purchased from the local market.

2.2. Biomass, enzymes, and yeast

Corn cobs were collected in the urban region of Natal, a city in Northeast Brazil. The corn cobs were initially broken using a hammer and then milled in a TE-680 knife mill (TECNAL, Brazil). The corncob samples were sieved through a 20-mesh sieve so that the retained fraction (> 20 mesh, 0.85–1.2 mm) and the passing fraction (< 20 mesh, 0.3–0.7 mm) were collected. For chemical composition and crystallinity analyses, both samples were subsequently milled to reach the appropriate particle size of 48 mesh.

The cellulolytic enzymes used in the present study are from the Cellic Ctec2 cocktail (or cellulase blend, number, Sigma Aldrich). The enzyme cocktail showed a cellulase activity concentration of 160 filter paper units (FPU) per mL. The Saccharomyces cerevisiae PE-2 strain was kindly provided by the Institute for Biotechnology and Bioengineering at the University of Minho. The strain was kept in the refrigerator (-4°C) in Petri dishes with potato-dextrose-agar medium for subsequent inoculation in fermentation experiments.

2.3. Acid pretreatment

Samples with 50 g of corn cob (either < 20 mesh or > 20 mesh) were mixed with 500 mL of 1% (w/v) sulfuric acid in 1000-mL Erlenmeyer flasks. The Erlenmeyer flasks were placed in a vertical autoclave AV (Phoenix, Brazil) at 121°C for a heating time of 20 min and a cooling time of 25 min. After cooling the Erlenmeyer flask, the mixture was cloth-filtered, and the solids were thoroughly washed with tap water. Then, the washed solids were dried in an air circulation oven (TECNAL, Brazil) for 24 h and stored in plastic pots for later use in enzymatic hydrolysis and fermentation.

Untreated and pretreated corncob samples were characterized by chemical composition and crystallinity. The values of cellulose, hemicellulose, Klason lignin, extractives, and ash were determined by NREL – National Renewable Energy Laboratory protocols [25–27]. Crystallinity was determined in an XRD-6000 diffractometer with Kα radiation, voltage of 40 kV, and electric current of 30 mA. The crystallinity indices (CrI) were calculated using Eq. 1, in which I₀₀₂ is the intensity of the crystalline plane 002 (2θ = ~22.6°) and I_am is the intensity of the valley between the peaks of planes 002 and 001 (2θ = ~18.7°).

$$CrI\left(\text{\%}\right)=\left(\frac{{I}_{002}-{I}_{am}}{{I}_{002}}\right)\times 100$$

2.4. Enzymatic hydrolysis

Enzymatic hydrolysis experiments of corn cob were carried out (duplicate) in 25-mL Erlenmeyer flasks containing sodium citrate buffer (50 mM, pH 4.8), Cellic Ctec2 cellulases and 0.05% (w/v) sodium azide. The Erlenmeyer flasks were placed in a shaker at 50°C and, after 48 h, the supernatants were collected after centrifugation with a centrifuge SL-700 (Solab, Brazil). The supernatants were subsequently analyzed for glucose and xylose using high-performance liquid chromatography (HPLC) (See section 2.8). Initially, corn cob samples with > 20 mesh and < 20 mesh were used to investigate the sugar release under cellulase dosages of 15, 10, and 5 FPU/g. The solid loading was set at 5% (w/v) solids. After screening the particle size and cellulase dosage, the solid loading was changed to higher values: 10% and 20% (w/v) solids. Experiments were carried out at 20% (w/v) solids with the abrupt and gradual addition of pretreated corncob into the reaction medium. The gradual addition of cob, i.e., corresponding to 10% (w/v) solids, occurred 24 h after the start of enzymatic hydrolysis. PEG 1500 was also added to boost enzymatic digestibility and the chemical was investigated at 2% (w/w of corncob).

2.5. Semi-simultaneous saccharification and fermentation

Ethanol production from pretreated corncobs was carried out (in duplicate) using semi-simultaneous saccharification and fermentation (SSSF). The S. cerevisiae PE2 inoculum was prepared from cultivation in a nutrient medium (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract, pH 4.8) for 24 h, and the cells were resuspended in citrate buffer (50 mM, pH 4.8). The mixture of corncob (5%-20%, w/v), cellulases, and sodium citrate buffer (50 mM, pH 4.8; without and with PEG 1500) was prepared in 25-mL Erlenmeyer flasks. Then, the Erlenmeyer flasks were placed in a shaker at 50°C and, after 12 h, S. cerevisiae cells were added to reach the final concentration of 1×10⁸ cells per mL. The Erlenmeyer flasks were incubated in a shaker at 40°C for 36 h. After incubation, the supernatants were collected via centrifugation and analyzed by HPLC (See section). Solid SSSF residue was collected, washed three times with deionized water, and dried in an air circulation oven (TECNAL, Brazil) at 60°C for 24 h.

2.6. Lignosulfonate from fermentation residue

The production of lignosulfonate from solid SSSF residue was carried out based on the procedures described by Ying et al. [28]. In a three-neck reactor, 1 g of SSSF residue was previously dissolved in the mixture of 97 mL of 1% (w/v) sodium hydroxide and 3 mL formaldehyde at 70°C for 1 h. Then, 100 mL of 20 g/L sodium bisulfite were added to the reactor and the reaction was kept under stirring at 70°C for 3 h. After completion of the reaction, the supernatant was allowed to reach room temperature and then placed in dialysis bags with a 1 kDa cut-off (Sigma-Aldrich). Water change was performed for the first 6 h up to 24 h and then changed every 24 h for another two days. The contents of the dialysis bag were transferred to plates and then dried in an TE394/I air circulation oven (TECNAL, Brazil) for 24 h.

The sulfomethylation product was characterized by a Fourier Transform Infrared Spectrometer (FTIR) using alkaline lignin from corncob as a reference. Alkaline lignin from corn cobs was prepared according to Padilha et al. [29]. FTIR analysis was performed in an IRAffinity-1 spectrometer (Shimadzu, Japan) in the 400 to 4000 cm^− 1 range with a resolution of 4 cm^− 1. The action of lignosulfonate as a surfactant was also investigated (triplicate). Two-phase water-oil systems were prepared with hydrophobic solvents, including toluene, n-octanol, and soybean oil. The total volume of the systems was 2 mL with a 1:1 volumetric ratio between phases. In these experiments, the concentration of lignosulfonate was investigated in the range of 0.5 to 2.0 g/L. The systems were placed under vigorous agitation for 15 s, and the droplet size and emulsification index responses at 24 h (E₂₄) were evaluated. The E₂₄ response was calculated as the ratio of the emulsified layer's height to the system's total height.

2.7. Phenolic acids from fermentation residue

The release of phenolic acids linked to the lignocellulosic matrix was done through alkaline hydrolysis. In test tubes, 20 mg of SSSF residue were mixed with 2 mL of reagent solution containing 4% (w/v) sodium hydroxide and hydrogen peroxide in percentages of 0%, 2%, and 5% (v/v). After 4 h of incubation under 80°C, the test tubes were left at room temperature, and then 2 mL of 1.5 M sulfuric acid was added to the room temperature. Lignin and solid residues were removed after centrifugation and phenolic acids in the supernatants were quantified via HPLC. The yield of phenolic acids was calculated in Eq. 2 from the ratio of phenolic acid mass generated to the raw material's initial mass.

$$Yield \left(\%\right)=\frac{{C}_{i}\times {V}_{R}\times DF}{m}$$

The term C_i corresponds to the phenolic acid concentration, VR is the reaction volume of alkaline hydrolysis, DF is the dilution factor, and m is the mass of corn cob used in the experiment.

2.8. HPLC analysis

HPLC analysis was performed in a Shimadzu platform (Shimadzu, Japan). The quantification of sugars (glucose, xylose, and arabinose) and carboxylic acids (formic acid, acetic acid, and levulinic acid) was carried out using a Shim-pack SCR101-H column (7.9 × 300 mm) with a mobile phase of 5 mM sulfuric acid at flow of 0.6 mL/min and a temperature of 50°C in the oven. The concentration of p-coumaric acid and ferulic acid, as well as furans (hydroxymethylfurfural and furfural) were quantified in Shim-pack CLC ODS C18 (4.6 × 150 mm) using a temperature of 35°C and a mixture of 1% (v/v) acetic acid-acetonitrile (ACN) as mobile phase. The mobile phase was modulated as follows: 0% ACN to 30% ACN up to 15 min, 30% ACN to 100% ACN up to 18 min, and 100% ACN to 0% ACN up to 20 min. Then, the mobile phase was kept without ACN for up to 32 min.

2.9. Statistical analysis

Tukey’s tests evaluated the significance of factor effects in enzymatic hydrolysis, fermentation, emulsification index, and alkaline hydrolysis experiments. The analyses were performed using the Statistica 7.0 software (StatSoft, USA) at a confidence level of 95% (p < 0.05).

3.1. Production of sugars and ethanol from corncob

Table 1 summarizes the chemical composition of the untreated and pretreated corncob samples. In general, the chemical compositions of the untreated corncob samples were similar. This behavior was expected since milling in a knife mill does not imply structural changes to the lignocellulosic matrix. Similar reports are shown in Liu et al. [30]. Regardless of the initial particle size, acid pretreatment effectively removed hemicellulose. In corncobs with an initial size of < 20 mesh, hemicellulose decreased from 34.37–7.87% after pretreatment. The hemicellulose reduction was from 27.63–8.12% in the pretreated sample > 20 mesh. Hemicellulose is an amorphous polysaccharide with glycosidic bonds susceptible to acid attack at mild temperatures (> 120°C), generating pentoses and organic acids in the liquid phase. The ash was also sensitive to acid pretreatment, reducing values below 1.2%. Alkaline metals (such as Na and K cations) and alkaline earth metals (such as Mg and Ca cations) are present in the ash of lignocellulosic biomasses, and they are easily removed in an acidic environment [31]. As a result, there was a proportional increase in the other components in the present study. Klason lignin values increased slightly in the pretreated corn cob from 14.54–24.74% in the > 20 mesh sample and 17.23–20.72%. Based on Schutyser et al. [32] and He et al. [33], using autoclave and acid was probably able to break down the lignin, but it eventually repolymerizes and deposits on the surface of the pretreated biomass. In turn, cellulose contents in pretreated samples increased almost twice compared to cellulose values in untreated samples. Enriching cellulose in biomass to levels such as ~ 60% is attractive for achieving high glucose concentrations in enzymatic hydrolysis.

The application of pretreatment generated solid yields of ~ 50%, which can be explained by the dissolution of hemicellulose and loss of ash in an acidic environment. In turn, the initial size of the untreated cob had little effect on this metric. The solid yield with corncob < 20 mesh was 51.4%, while the solid yield with corncob > 20 mesh was 53.5%. The reduction in particle size is generally favorable for solid-liquid systems with a reaction due to increased surface area. Here, the slight drop in yield suggests pretreatment success but implies a smaller quantity of solids destined to generate sugars and ethanol.

The crystallinity values reinforce the effectiveness of the acid pretreatment. For samples > 20 mesh, the ICr values of untreated and pretreated corncobs were 42.1% and 44.8%, respectively. The untreated sample < 20 mesh presented an ICr of 39.3%, and the pretreated sample < 20 mesh presented an ICr of 45.1%. This behavior can be explained by the proportional increase in cellulose in the corn cob after pretreatment. Costa Filho et al. [4] and Nogueira et al. [11] also recorded a significant increase in corncob crystallinity after acid pretreatments.

Table 1

Solid yield, chemical composition, and crystallinity of untreated and acid pretreated corncob samples
Samples	Solid yield (%)	Cellulose (%)	Hemicellulose (%)	Klason lignin (%)	Extractives (%)	Ash (%)	ICr (%)
Untreated CC (< 20 mesh)	-	32.15 ± 2.15	34.37 ± 1.13	17.23 ± 1.67	9.23 ± 0.63	2.47 ± 0.1	39.3
Untreated CC (> 20 mesh)	-	30.28 ± 0.97	27.63 ± 1.06	14.54 ± 0.16	11.17 ± 0.63	3.16 ± 0.1	42.1
Acid pretreated CC (< 20 mesh)	51.4	63.92 ± 1.56	7.87 ± 0.24	20.72 ± 1.91	11.46 ± 1.91	0.48 ± 0.00	45.1
Acid pretreated CC (> 20 mesh)	53.5	61.96 ± 3.78	8.12 ± 0.51	24.74 ± 3.40	11.00 ± 0.84	1.2 ± 0.00	44.8

Figure 2 shows the sugar release in the enzymatic hydrolysis of untreated and pretreated corncobs under different enzyme dosages. Unlike pretreatment experiments, particle size was important at this stage. For untreated samples, reducing particle size from > 20 mesh to < 20 mesh increased glucose release from 4.44 to 6.40 g/L. The difference between the responses is 44.1%, although it is not statistically significant. Similar behavior was observed for the release of xylose in these samples, increasing from 1.77 to 2.66 g/L. After pretreatment, the corncob became less recalcitrant, and its polysaccharides were more accessible to enzymatic attack, which explains the increased production of glucose and xylose. The increase in xylose release is curious since the pretreated sample has a lower hemicellulose content than the untreated sample. Glucose release from pretreated corncob reached 18.79 g/L in the < 20 mesh sample and 18.90 g/L in the > 20 mesh sample using 15 FPU/g. However, the granulometry was again impactful when lower dosages of cellulases were applied. Using 5 FPU/g (lowest dosage of cellulases), the pretreated sample < 20 mesh released 15.23 g/L, while the sample released only 11.10 g/L.

When cellulases are in excess, as possibly in the 15 FPU/g condition, mass transfer aspects can be left aside in our study. Even because the experiments were conducted at 50°C (the optimum cellulase temperature) and the final incubation was 48 h. However, difficulties linked to using larger particles, such as low surface area and higher viscosity, become more relevant when there is a lack of cellulases in the medium (5 FPU/g). In Lu et al. [34], corn stover samples with average sizes of 286.7 and 75.2 µm showed 16.8% and 18.1% glucose release using 5 FPU/g. Using rice straw as substrate, Kapoor et al. [35] reported identical glucose release (~ 88 g/L) in experiments with a particle size of 5 mm and 5 FPU/g and a particle size of 20 mm and 10 FPU/g.

To maximize the sugar release, pretreated corncob with < 20 mesh was chosen in the following steps. Choosing samples with > 20 mesh would also be interesting since they consumed less energy for comminution, but we adopted another criterion. About cellulases, the dosage of 5 FPU/g was chosen to maintain the glucose produced at the same level, consuming three times less enzyme, an expensive input for second-generation bioethanol technology.

Based on the previous graph (Fig. 2), glucose release was good from pretreated corn cob with < 20 mesh using 5 FPU/g, but we suspected it could be even higher. Higher ethanol titers are required to ensure the economic viability of second-generation bioethanol technology, which requires higher solid loadings. Solid loading of 5% (w/v) is interesting for screening operational conditions; however, the technology must be tested at solid loadings superior to 15% (w/v). The cellulosic conversion in experiments with 5% (w/v) was reasonable (~ 44.3%), but it indicates that more cellulose could be digested. To increase the sugars generated, PEG 1500 was added to mitigate non-productive adsorption in enzymatic hydrolysis and its results are presented in Fig. 3.

Increasing the solid loadings increased the release of glucose and xylose. In experiments without PEG 1500, glucose release increased from 15.23 g/L at 5% (w/v) solids to 23.61 g/L at 10% (w/v) solids. The same goes for the release of xylose, which increased from 3.32 to 4.07 g/L. This behavior is directly associated with raw material availability in the reaction environment. When more solids were added, there was a slight increase in glucose release with 20% (w/v) solids (26.2 g/L) and a substantial increase in glucose production with the gradual addition of solids (10% + 10%, w /v). Although these conditions have the same final amount of solids, gradually adding biomass causes less stress. High amounts of solids increase the viscosity of the environment and reduce free water, compromising the performance of enzymes. The practice of gradually adding biomass in enzymatic hydrolysis was successful in the work of Gao et al. [36], Mukasekuru et al. [37], and Nogueira et al. [38].

The addition of PEG 1500 also played a critical role in enzymatic hydrolysis. Enzymatic hydrolysis with 5% (w/v) solids and PEG 1500 achieved 24.39 g/L glucose and 4.27 g/L xylose, a performance increase of 60% and 29% compared to the same experiment without additive. These results were superior to those obtained at 10% (w/v) solids without PEG 1500. The cellulose conversion in enzymatic hydrolysis with 5% (w/v) solids was 68.7%, a result comparable to other papers that used low dosage in the enzymatic hydrolysis of cob [4, 39, 40]. Using the gradual addition of biomass, the experiment with PEG 1500 achieved a glucose release of 56.73 g/L, the best volumetric production result among the experiments. A disclaimer is necessary when compared to our previous study. Nogueira et al. [11] investigated the effects of PEG on ethanol production from corncobs. In this study, unlike what was presented here, adding PEG is not relevant to boosting enzymatic digestibility. These discrepancies may be linked to enzyme dosage. The paper by Nogueira et al. [11] operated at 20 FPU/g, four times higher than the enzyme dosage used in the present study. The excess enzymes likely masked any effects of non-productive adsorption onto residual lignin and deactivation of cellulase at the air-liquid interface.

The addition of PEG 1500 and changes in solids load were investigated in saccharification and semi-simultaneous fermentation (SSSF), as seen in Fig. 4. Tests with saccharification and simultaneous fermentation (SSF) were also carried out, but ethanol production was negligible. (data not shown). The low enzyme dosage of 5 FPU/g in SSF could not generate sugars to maintain yeast cells; therefore, ethanol production was compromised. This hypothesis is corroborated by HPLC analysis that showed high glucose concentrations at the end of the experiment. As expected, ethanol production increased with the increase in solid loadings. Ethanol values reached 3.00, 6.67, 20.18 and 26.71 g/L without PEG 1500 with 5%, 10%, 20% (w/v) abrupt addition, and 20% (w/v) gradual addition, respectively. PEG 1500 boosted ethanol production in the SSSF, mainly in the condition with 10% (w/v) solids (17.73 g/L and ethanol yield of 48.8%). Conceição Gomes et al. [41] reported the benefits of adding PEG in SSSF experiments. Statistical differences were not observed in experiments with 20% (w/v) solids. Experiments with abrupt addition without and with PEG 1500 obtained 20.18 and 21.65 g/L, respectively. In experiments with gradual addition, PEG 1500 promoted an advantage in average ethanol production (31.64 g/L vs. 26.71 g/L) compared to the additive-free condition. The enzyme dosage of 5 FPU/g is low; therefore, reducing the temperature to 40°C in fermentation step and restricting free water reduced the efficiency of cellulases. Probably, better ethanol results at high solid loadings would be achieved by increasing the cellulase dosage (perhaps increasing it to 7.5 or 10 FPU/g). Still, the present study results are surprising compared to other reports with corncob. According to Paschos et al. [42], 30–40 g/L ethanol titers are already capable of ensuring the economic viability of second-generation bioethanol technology.

3.2. Production and application of lignosulfonate

Figure 5 shows images of the lignosulfonate produced. Lignosulfonate has a light brown hue, typical of lignin, and has broad solubility in water. The lignosulfonate yield was 23.3%, considered low compared to studies on surfactant preparation based on isolated lignin. However, it is worth highlighting that the fermentation residue still contains relevant levels of cellulose (~ 25%), which could still be linked to lignin.

Figure 6 presents the FTIR spectra of lignosulfonate and alkaline lignin. Similarities between the spectra are evident since aromatic structures form both substances. However, some differences must be highlighted. Lignosulfonate reduced the peaks at 1685 and 1150 cm^− 1, corresponding to the C = O vibration in non-conjugated moieties [43, 44]. Lignosulfonate presented a peak in the region of 1373 cm^− 1, attributed to the vibration of in-plane deformation of phenolic hydroxyl, according to Sameni et al. [45]. These differences may arise from the different processing to which each lignin was exposed. The fermentation residue originated from an acid pretreatment, in which the lignin structure is disrupted and repolymerized, which hindered the subsequent extraction/functionalization of the biopolymer during lignosulfonate synthesis.

The formation of lignosulfonate was confirmed by the presence of the peak located at 1208 cm^− 1. According to Wibowo et al. [46], this peak is attributed to the vibration of S = O bonds, which results from the attachment of the sulfomethyl group in the ortho position of phenolic groups. The lack of a peak in lignosulfonate at 573 cm^− 1 may also reflect the success of functionalization. Stretching vibrations of aromatic -CH are assigned at wavenumbers in the region of 618 − 542 cm^− 1 [47].

The emulsification index values of the different hydrophobic solvent/water systems are shown in Table 2. The height of the emulsified layer was directly affected by the lignosulfonate concentration. Just like any surfactant, lignosulfonate has hydrophobic groups (in this case, aromatic groups) and hydrophilic groups (i.e., sulfonate and hydroxyl groups), and their increase guarantees greater dispersion of hydrophobic solvent in the aqueous phase. Remarkable results were achieved in the n-octanol/water system, in which the E₂₄ value reached up to 33.33% using 1 g/L lignosulfonate. The positive effects of increasing concentration on the stabilization of n-octanol/water systems are also shown in Fig. 7. Most droplets generated (q = 89%) were smaller than 30 µm using 1 g/L lignosulfonate. Droplets smaller than 30 µm do not correspond to even 30% (q = 30%) of the droplet population in the n-octanol/water system without lignosulfonate. According to Uchida et al. [48], smaller droplets are less susceptible to instability phenomena.

The worst performance of lignosulfonate was observed in the toluene/water system. This result was intriguing since toluene is a planar molecule and π-π interactions between it and lignin were expected to drive stabilization. In Padilha et al. [29], the authors achieved good emulsification rates of around 68% using 1 g/L alkaline lignin nanoparticles as stabilizers.

Comparing our results with other papers involving lignosulfonate was a complicated task. Although lignosulfonates have been used for decades as a dispersant, data in the literature still need to be provided regarding emulsification index experiments. To our knowledge, the only work that investigated the role of lignosulfonate in stabilizing oil/water systems was Ruwoldt et al. [49], but another hydrophobic solvent (xylene) was used. There are other more popular purposes for lignosulfonates, such as cement pastes or biotechnological applications [50–52]. However, this type of experiment is quick and practical to confirm the formation of lignosulfonate. Compared to commercial surfactants, such as sodium dodecyl sulfate (SDS) [53], the performance of lignosulfonate was low and raises the possibility of improving surfactant synthesis in future work. Extraction of alkaline lignin from fermentation residue and oxidation could disrupt aromatic rings and increase functionalization sites.

Table 2

Emulsification indexes of oil/water systems (1:1, v/v) with lignosulfonate (LS) obtained from corncob fermentation residue.
Systems	Emulsification index (E₂₄, %)
Systems	0.2 g/L LS	0.5 g/L LS	1.0 g/L LS
Toluene/Water	9.17 ± 1.67^c	9.33 ± 1.88^c	18.99 ± 0.34^b
n-octanol/Water	8.00 ± 0.00^c	28.56 ± 1.62^a	33.33 ± 1.81^a
Soybean oil/Water	8.94 ± 2.16^c	9.33 ± 1.89^c	30.77 ± 0.00^a

3.3. Alkaline hydrolysis performance

In addition to lignosulfonate, phenolic acids were produced from the fermentation residue and the yield results are presented in Table 3. The production of p-coumaric acid was prioritized over ferulic acid in all conditions investigated. Using only sodium hydroxide in the liquid phase, the yield of p-coumaric acid reached 2770.7 mg/100 g, while ferulic acid reached 496.0 mg/100 g. Both responses increased with the addition of hydrogen peroxide. The p-coumaric acid values were 5178.6 and 8045.3 mg for every 100 g of fermentation residue in alkaline hydrolysis with 2% and 5% (v/v) hydrogen peroxide, respectively. Ferulic acid yields were 891.3 and 1429.4 mg/100 g using 2% and 5% (v/v) hydrogen peroxide, respectively.

Sodium hydroxide breaks ether and ester bonds in biomass, releasing phenolic acids into the liquid phase during alkaline hydrolysis. Hydrogen peroxide releases active oxygen to break C-C bonds in the lignin structure, an additional mechanism for generating phenolic acids [32]. Ferulic acid is linked to lignin and hemicellulose [54], which explains the loss of the phenolic acid in the acid pretreatment step. In a study on the generation of phenolic acids from corn fiber, Valério et al. [55] showed a loss of almost 20% of ferulic acid in the pretreatment stage.

p-Coumaric acid is a constituent of lignin and its high yield in the present study is due to the lignin accumulation in the fermentation residue. High yields of p-coumaric acid are reported in the literature, such as in Mussatto et al. [23] (922 mg/100 g) or Timokhin et al. [22] (4800 mg/100 g); however, these values are still several times lower than the results of the present study. The p-coumaric acid generated is also in high purity, as seen from the HPLC chromatogram (Fig. 8). Given these results, obtaining p-coumaric acid from fermentation residue must be praised. In addition to its antioxidant properties offered via ingestion, p-coumaric acid is used as a precursor for new functional materials, including polymers, adhesives, pharmaceuticals, and nutritional products [22]. It is also noteworthy that p-coumaric acid is estimated at $50/kg, at least ten times more expensive than ethanol [56, 57].

Table 3

Yields of phenolic acids generated from alkaline hydrolysis of solid saccharification residue and semi-simultaneous fermentation. The experiments were carried out with 1% (w/v) solids, a temperature of 80°C, and incubation for 4 h.
Conditions	p-coumaric acid (mg/100g)	Ferulic acid (mg/100 g)
4% (w/v) Sodium hydroxide	2,770.7 ± 201.7^c	496.0 ± 28.9^c
4% (w/v) Sodium hydroxide + 2% (w/v) Hydrogen peroxide	5,178.6 ± 131.9^b	891.3 ± 33.3^b
4% (w/v) Sodium hydroxide + 5% (w/v) Hydrogen peroxide	8,045.3 ± 442.7^a	1,429.4 ± 64.3^a

The conversion of corncob into multiple value-added products has been successfully executed. Acid pretreatment with sulfuric acid effectively enriched biomass with cellulose in samples with different particle sizes (smaller than 20 mesh and larger than 20 mesh). The addition of 2% (w/v) PEG 1500 boosted the release of sugars by 60.1–5% (w/v) solids and by 57.1–20% (w/v) solids, a condition with free water restriction. PEG 1500 also supported ethanol production via corncob SSSF, especially in the 10% (w/v) solids condition. Enzyme dosages above 5 FPU/g are recommended to increase ethanol titers with high solid loadings. Lignosulfonate was successfully generated from fermentation residue and it was able to stabilize oil/water systems, mainly the n-octanol/water system. The release of p-coumaric acid and ferulic acid was directly associated with the dosage of hydrogen peroxide. The p-coumaric acid yields found in the present study surpass results reported in the literature for other biomasses and reinforce the idea of valuing fermentation residues. Therefore, the scheme proposed from corn cobs proved to be interesting as it generated three different products with commercial appeal and gave hope for the consolidation of second-generation bioethanol through biorefineries.

Ethical Approval

This article does not contain any studies with humans or animals performed by any of the authors.

Competing interests

The authors declare there are no conflicts of interest.

Author´s contribution

Juli Maduzzi: Conceptualization, Experiments execution. Habila Thomas: Experiments execution. José Dário Fidelis: Experiments execution José Valderisso Carvalho: Experiments execution. Elano Silva: Experiments execution, Software, Reviewing and Editing; José Daladiê Costa Filho: Writing- Original draft preparation. José Demétrio Cavalcante: Writing- Original draft preparation. Everaldo Santos: Supervision, Resources. Domingos Souza: Supervision, Resources. Carlos Padilha: Conceptualization, Experiments execution, Writing- Original draft preparation, Formal analysis, Supervision

Fundings

CAPES (Coordination for the Improvement of Higher Education Personnel – Finance code 001) and CNPq (National Council for Scientific and Technological Development).

Availability of data and materials

Data and materials are available on request.

Acknowledgments

The authors gratefully acknowledge the Brazilian research funding agencies CNPq and CAPES for financial support.

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Coproduction of cellulosic ethanol, lignosulfonate, and phenolic acids from an integrated corn cob valorization scheme

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Abstract

Figures

1- Introduction

2- Materials and methods

2.1. Chemicals

2.2. Biomass, enzymes, and yeast

2.3. Acid pretreatment

2.4. Enzymatic hydrolysis

2.5. Semi-simultaneous saccharification and fermentation

2.6. Lignosulfonate from fermentation residue

2.7. Phenolic acids from fermentation residue

2.8. HPLC analysis

2.9. Statistical analysis

3- Results and discussions

3.1. Production of sugars and ethanol from corncob

3.2. Production and application of lignosulfonate

3.3. Alkaline hydrolysis performance

4- Conclusions

Declarations

References

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Journal Publication

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