Sequential Enzymatic and Mild-Acid Hydrolysis of By-Product of Carrageenan Process from Kappaphycus alvarezii

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Sequential Enzymatic and Mild-Acid Hydrolysis of By-Product of Carrageenan Process from Kappaphycus alvarezii

Fernando Roberto Paz-Cedeno¹&Eddyn Gabriel Solórzano-Chávez¹&Levi Ezequiel de Oliveira²&Valéria Cress Gelli³&

Rubens Monti⁴&Samuel Conceição de Oliveira¹&Fernando Masarin¹

#Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract

Kappaphycus alvareziiis a red macroalgae widely used to produce carrageenan. The carrageenan processing produces a by- product rich in glucan which has been reported as easily hydrolyzed with enzymes, but the hydrolysate forms a gel at usual fermentation temperatures. The purpose of this study was to evaluate the enzymatic hydrolysis integrated with a mild-acid treatment of the by-product to obtain a hydrolysate rich in monomeric sugars. Using an enzyme load of 10 FPU g⁻¹of by- product, close to 100 and 14.7% of glucan and galactan conversion were reached, respectively. Increasing the enzyme load to 100 FPU g⁻¹raised the galactan conversion to 30%. The mild-acid treatment after enzymatic hydrolysis was satisfactory, increasing the glucose and galactose concentrations, without producing significant amounts of fermentation inhibitors and avoiding the formation of a gel structure. The statistical analysis showed that the main effects on the response were negative for the three independent variables, meaning that the selectivity (S) becomes lower when experimental conditions at the higher levels are used (longer time, higher temperature, and acid concentration). Therefore, the integrated enzymatic and acid hydrolysis of the by-product becomes a promising technological route to produce monomeric sugars for bioethanol or fine chemical production.

Keywords Kappaphycus alvarezii. By-product . Carrageenan . Chemical composition . Enzymatic and acid hydrolysis

Highlights

•A by-product of carrageenan process fromK. alvareziiwas obtained.

•The chemical composition of the by-product was determined.

•Integrated enzymatic and mild-acid hydrolysis was performed.

•The mild-acid hydrolysis was evaluated using a full factorial design.

•A hydrolysate was obtained without formation of fermentation inhibitors.

Electronic supplementary materialThe online version of this article (https://doi.org/10.1007/s12155-019-09968-7) contains supplementary material, which is available to authorized users.

* Fernando Masarin fernando.masarin@unesp.br Fernando Roberto Paz-Cedeno fernando.paz@unesp.br Eddyn Gabriel Solórzano-Chávez eddynsch04@hotmail.com Levi Ezequiel de Oliveira levi_ezequiel@yahoo.com.br Valéria Cress Gelli

valeriagelli@pesca.sp.gov.br Rubens Monti

rubens.monti@unesp.br

Samuel Conceição de Oliveira samuel.oliveira@unesp.br

1 School of Pharmaceutical Sciences (FCF), Department of Bioprocesses and Biotechnology, UNESP—São Paulo State University, Araraquara, SP 14800-903, Brazil

2 Lorena School of Engineering (EEL), Department of Chemical Engineering, USP—University of São Paulo, Lorena, SP, Brazil

3 North Coast Research and Development Center, Secretariat of Agriculture and Supply of the State of São Paulo, Fisheries Institute (IP), São Paulo, Brazil

4 School of Pharmaceutical Sciences (FCF), Department Food and Nutrition, UNESP—São Paulo State University,

Araraquara, SP 14800-903, Brazil https://doi.org/10.1007/s12155-019-09968-7

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Introduction

Biofuels represent a topic of global interest due to the depen- dence of petroleum derivatives on energy production and its consequences on global climatic changes. According to the Renewable Fuels Association, the main producers of ethanol in 2017 were United States (62.2 billion L) and Brazil (27.4 billion L), accounting together for 84% of the world production [1].

Currently, bioethanol is produced, mainly, from sugarcane, corn starch, and lignocellulosic materials. However, lignocellulosic materials are very recalcitrant to the enzymatic hydrolysis of polysaccharides, which is an important step in bioethanol production, requiring a pretreatment step of the material and subsequent hydrolysis for conversions above 30% [2–5]. Another renewable source that can be used to produce bioethanol is macroalgae. They are terrestrial and aquatic plants, and many species contain high percentages of carbohydrates and low percentage of lignin [6–10]. Due to the low lignin content, polysaccharides are more accessible to enzymatic treatments, which makes macroalgae less recalcitrant than terrestrial plants [11–13].

Kappaphycus alvareziiis a seaweed of high commercial value, grown as a raw material for the industrial production of carrageenan [13, 14]. Carrageenan accounted for an annual global market of U$$ 600–700 million in 2016 with a projected average growth of approximately 3%

[1 5] . B r a z i l p r o d u c e d 7 0 0 t o n o f b i o m a s s f r o m K. alvarezii, but this production was not enough to satisfy the national economic demand [16]. The main components of K. alvarezii are carbohydrates (mainly galactan and glucan), reaching values greater than 50%. Among other components of K. alvarezii are minerals, sulfate groups, and proteins [11–13,17]. In the production process of refined carrageenan, a by-product composed mainly of glucans is produced. This by-product is not suitable for human consumption; however, it is commercialized as semi-refined carrageenan and used in feed manufacturing [11–1 3, 1 8] . T h e a n n u a l w o r l d p r o d u c t i o n o f Kappaphycus alvarezii in 2016 was 1.5 million of tons [16]. The yield of by-product in the carrageenan refining process from Kappaphycus alvarezii is about 25% [11, 12]. Thus, the estimated quantity of the by-product is 0.38 million of tons per year.

The glucan fraction of this by-product has previously been reported as easily hydrolyzed using commercial enzymes containing cellulase activity. Nevertheless, the galactan fraction of the by-product is poorly hydrolyzed by commercial cellulolytic enzymes [11,12]. Also, the obtained hydrolysate forms a gel at 30 °C, which causes a problem for a subsequent fermentation because it precludes action of yeast.

For an efficient hydrolysis, the synergistic action of the following three main groups of enzymes is required: endo-

1,4-β-D-glucanase (EC 3.2.1.4), exo-1,4-β-D-glucanases (EC 3.2.1.91), andβ-D-glucosidases (EC 3.2.1.21). The three groups catalyze the hydrolysis ofβ-1,4 glycosidic bonds and act synergistically, creating new sites for hydrolysis [19,20].

Complex carbohydrates also can be hydrolyzed with acid treatment. During an acid hydrolysis of carbohydrates, sugar yield and fermentation inhibitor production mainly depend on three major factors, which are temperature, acid concentration, and reaction time. Usually, severe reaction conditions lead to more fermentation inhibitor compound generation, such as hydroxymethylfurfural (HMF), formed from the dehydration of hexoses [21–23]. Thus, submitting the enzymatic hydrolysate to an acid hydrolysis treatment in mild conditions is an interesting strategy to avoid the gelation of the hydrolysate and increase the concentration of monomeric sugars, but without producing fermentation inhibitors, like HMF. This hydrolysate has the potential to be used in the production of bioethanol or high-value products, as acetic, butyric, propionic, succinic, and itaconic acids [24–29].

In this context, the purpose of this study was to evaluate the enzymatic hydrolysis followed by acid hydrolysis under mild conditions of a by-product generated in the carrageenan process fromK. alvareziito obtain a hydrolysate rich in monomeric sugars with low or none fermentation inhibitors and avoiding the formation of a gel structure at fermentation temperature.

Material and Methods

Enzymes and Chemicals

The commercial enzyme mixtures Cellic CTec II, Celluclast, Pectinex Smash XXL, and Pectinex Ultra Clear were donated by Novozymes. Cellulases (from Trichoderma reesei), pectinase (from Aspergillus niger), carrageenan, gum Arabic, 3–5 dinitrosalicylic acid (DNS), sulfuric acid, and HPLC standards were purchased from Sigma-Aldrich (St.

Louis, USA). All other chemicals were of analytical grade or better. All solutions were prepared with ultrapure water (Millipore, Milli-Q®).

By-Product Obtainment

In this study, equal quantities of biomass from morphotypes with varied pigmentation (red, green, brown) and a strain de- rived from the germination of tetraspores (known as G11) were used [30–33]. The strains were supplied for the Fisheries Institute (Ubatuba, SP, Brazil). Figure 1 shows a flowchart of by-product obtainment and hydrolysis steps.

A sample of 35 g (dry weight) of the biomass was washed with 1 L of distilled water under stirring in a beaker for 45 min and dried at 25 °C. This material was

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termed asBuntreated biomass.^Cold alkali transformation was performed by soaking 8 g of untreated biomass in 100 mL of 6% KOH solution (w/v) for 24 h at 25 °C.

After this treatment, the biomass was washed with distilled water at least 5 times and oven-dried at 40 °C until constant weight. This material was named Btreated biomass.^ The KOH treatment yields were calculated di- viding the final dry mass by the initial dry mass (Eq. S1).

The experiments were conducted in duplicate.

Subsequently, the treated biomass was milled and subjected to extraction with distilled water at a ratio of 1 g (dry weight) of material to 160 mL of water. This process was performed in a shaker at 65 °C under orbital agitation of 120 rpm for 2 h. The suspension generated was passed through a filter of nylon tissue. The part retained in the filter was named asBby-product,^and the part that passed through the filter was denominatedBrefined-carrageenan^

[11, 12, 33, 34]. All fractions were oven-dried at 40 °C until constant weight and milled and passed through a 0.84-mm screen. The yields of the by-product and refined carrageenan were calculated similarly to the yields of materials treated with KOH (Eq. S1), but in relation to both materials, treated with KOH and untreated biomass (dry weight), where, R = mass yield of by-product or refined carrageenan (%), Wi = initial dry mass (untreated or treated with KOH) (g), and Wf = final dry mass (by-product or refined carrageenan) (g). The experiments were performed in triplicate.

Chemical Composition

Carbohydrate and Organic Acid ContentsThe samples were hydrolyzed with 72% sulfuric acid (w/w) at 30 °C for 1 h and, then, with 4% sulfuric acid (w/v) at 121 °C for 1 h, as described in the literature [2,11,12]. Subsequently, the hydrolysate was filtered using a porous glass filter (Schott

#3, Germany). The material retained in the filters was dried, cooled, and weighed. This material corresponds to the insoluble aromatics. For detection of monomeric sugars (glucose and galactose) and decomposition products (formic and levulinic acid), the filtered hydrolysate was passed through a 0.45 μm membrane and a Sep-Pak C18cartridge. Compounds were, first, identified in a high- efficiency liquid chromatography (HPLC) system equipped with a HPX87P column (Bio-Rad Hercules, CA, USA) at 60 °C by elution with deionized water at a rate of 0.6 mL min⁻¹. The detection was performed using a refractive index detector (RID) at 60 °C (Shimadzu, model C-R7A). For quantification, samples were analyzed in a HPLC system equipped with a HPX87H column (Bio-Rad Hercules, CA, USA) at 60 °C by elution with 0.005 M H2SO4 at a rate of 0.6 mL min⁻¹ and detected using a refractive index detector (RID) at 60 °C (Shimadzu, model C-R7A).

Hydroxymethylfurfural Hydroxymethylfurfural (HMF) was quantified passing the filtrate through a 0.45μm membrane Fig. 1 Flowchart of by-product

obtainment and hydrolysis steps

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and, then, analyzed in a HPLC system (Flexar, Perkin-Elmer) equipped with a C18column (Hypersil, Thermo-Scientific) at 25 °C by elution with acetonitrile:water (1:8,v/v) containing 1% (v/v) of acetic acid at a rate of 0.8 mL min⁻¹. The detection was performed using an UV–Vis detector at 276 nm [11]. The mass percentages of the glucan and galactan fractions were calculated according to Eqs. S2 and S3, respectively.

Protein ContentA Kjeldahl digester was used to determine the total nitrogen of the samples. The obtained value was multi- plied for a nitrogen conversion factor of 4.92 to calculate the protein content [12,35,36].

Ash ContentThe ash content was determined according to the NREL standard [12,37]. For this purpose, approximately 1 g (dry weight) of sample was weighed, placed in porcelain cru- cible, and oxidized in a muffle furnace at 575 ± 25 °C, using a preset heating ramp. At the end of 3 h, the crucibles were cooled and weighed.

Lipid ContentFor lipid determination, 1 g (dry basis) of sample (only untreated biomass) was submitted to extraction with hexane (99%,v/v) for 8 h using a Soxhlet extractor. Then, the samples were dried at 25 °C and weighted. The difference of weight (dry basis) between extracted and non-extracted samples was considered as lipids [11,12,17]. This compound was not determined in the other fractions because the KOH treatment removes it from the biomass [11].

Sulfate Group Content The sulfate groups were quantified using modified spectrophotometric methods as described in the literature [11,12,33,38]. Briefly, 0.05 g (dry basis) of the milled sample was treated with 1 mL of 0.5 N HCl at 120 °C for 1 h. Next, distilled water was added to complete 10 mL and centrifuged at 7000 ×gfor 10 min. A total of 2 mL of the supernatant were recovered, added with 18 mL of distilled water, 2 mL of 0.5 N HCl, and stirred. Finally, 1 mL of BaCl2gelatin was added. The mixture was kept in stirring in a roller mixer for 30 min at 25 °C. Absorbance was taken at 550 nm (Ultrospc 3100 Pro, Amersham/ Biosciences). All the experiments for determination of chemical composition were conducted in triplicate.

Determination of the Proteins and Enzymatic Activity of Enzymatic Extracts

Total CellulasesTotal cellulase activity was determined following the methodology described by Ghose [39].

Briefly, 1 mL of 50 mM sodium acetate buffer pH 4.8 was placed in a tube, added with a strip of filter paper (1 × 6 cm; approximately 50 mg) and 0.5 mL of enzyme extract. The reaction was conducted at 50 °C for 60 min. Then, 3 mL of 3-5 dinitrosalicylic acid (DNS)

was added, and the mixture was boiled for exactly 5 min. Finally, 20 mL of distilled water was added to the tubes, and readings were taken in a spectrophotometer at 540 nm.

Galactanases For the galactanase activity, two substrate solutions were used separately, carrageenan (0.1%; w/v) and gum Arabic (0.5%; w/v) in sodium acetate buffer pH 5.5 [40]. Thus, 0.1 mL of enzyme extract and 0.9 mL of substrate solution (carrageenan or gum Arabic) were mixed and allowed to react for 60 min at 50 °C. Then, 1.5 mL of 3 mL of DNS was added to the mixture and boiled for 5 min. Spectrophotometer readings were taken at 540 nm.

ProteinsFor protein determination of commercial enzymatic extracts, the methodology described by Lowry modified by Hartree [41] was used. Accordingly, 0.1 mL of enzyme extract was mixed with 2.5 mL of a solution containing potassium sodium tartrate, sodium carbonate, sodium hydroxide, copper sulfate and allowed to react for 15 min. Then, 0.25 mL of Folin-Ciocalteu 1 N reagent was added. After 30 min, spectrophotometer readings were taken at 660 nm.

Enzymatic Hydrolysis

The obtained by-product was hydrolyzed using commercial enzyme extracts of cellulases (Cellic CTec II, Celluclast, Cellulases from Trichoderma reesei) and pectinases (Pectinase, Pectinex Smash XXL, Pectinex Ultra Clear). An enzymatic load of 10 and 100 FPU (cellulases) or 20 UI (pectinase) per gram of substrate was used. All reactions were performed at 2% (w/v) of consistency in 50 mM sodium acetate buffer, pH 4.8 under orbital agitation at 120 rpm at 45 °C for 72 h [11,12]. For the determination and quantification of sugars resulting from enzymatic hydrolysis, the supernatant was previously diluted and analyzed by HPLC (item Chemical Composition). The experiments were performed in triplicate. The data of chemical composition, the factors of enzymatic hydrolysis (0.9, for glucose and galactose), and the final volume of the reaction (5 mL) were used to calculate the percent conversion of glucan to glucose and galactan to galactose (Eq. S4).

The experimental glucose concentration data (g) from the enzymatic hydrolysis were fitted by a mathematical model (Eq. (1)) previously developed and validated for the enzymatic kinetics under study, which is based on the fact that glucose concentration grows over time and reaches an asymptotic final value [42,43].

g¼g_max 1−e^−kt

ð1Þ

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In Eq. (1),g = glucose concentration (g L⁻¹),gmax = asymptotic maximum value of glucose concentration predicted by the model (g L⁻¹),t= hydrolysis time (h),k= kinetic constant related to glucose accumulation (h⁻¹).

The estimation of the model parameters was performed in the Origin software (OriginPro 2017), obtaining the kinetic parametersgmax,k, andgmax∗k(maximum glucose formation rate) [42,44,45]). As a fair first approximation, the quality of fit of the nonlinear mathematical model was evaluated through coefficient of determination (R²). Data of maximum glucose concentration was evaluated using the Tukey’s test (GraphPad Instat software) with a significance level of 0.05 (confidence degree of 95%).

Acid Hydrolysis

For the acid hydrolysis, three enzymatic hydrolysates were used separately. They were obtained using the extract Cellic CTec II. The first one was using an enzymatic load of 100 FPU g⁻¹for 72 h, the second one using an enzymatic load of 100 FPU g⁻¹for 4 h, and the third one using an enzymatic load of 10 FPU g⁻¹for 4 h. A 2³full factorial design with three independent variables was used (temperature, time, and acid concentration), at two levels and three replicates at the center point (Table 5). Each hydrolysate was placed into Falcon tubes, and sulfuric acid was added at different concentrations to reach 0.5, 1.25, or 2% of final acid concentration respectively. The enzymes contained in the hydrolysates were inactivated at the moment the acid was added due to the decrease in pH to (at least) 1. The tubes were placed in a shaker at 120 rpm, and the temperature was set at 60, 70, and 80 °C, in accordance with the experiment programming.

The experimental planning matrix reporting the conditions of the factors evaluated in the acid hydrolysis (temperature, time, and acid concentration) is included in Table5.

The statistical analysis and experimental design data modeling were performed using theSTATISTICA 10 software, developed by StatSoft®.

Results and Discussion

Chemical Characterization of the Fractions

from the Process of Obtaining Refined Carrageenan

The biomass from the macroalgaeK. alvareziiwas submitted to a process to obtain refined carrageenan. The biomass processing generated three fractions, which are as follows: biomass treated with KOH, refined carrageenan, and a by-product. The refined carrageenan was obtained as a soft, soluble,

and translucent material, while the by-product was a solid, insoluble, and thick material (Fig.S1).

For the determination of the chemical composition of the fractions obtained in the present study, the acid hydrolysis methodology for lignocellulosic materials was used [2,11, 12]. After the acid hydrolysis steps, the monomeric sugars present in the hydrolysates were analyzed. In addition, the content of HMF, formic, and levulinic acids was determined.

These are degradation products formed from hexoses during the acid hydrolysis [17,46]. FigureS2shows chromatograms of analytical standards and the hydrolysate of by-product.

The amount of HMF produced with the acid hydrolysis of the untreated biomass was similar to that reported in the literature for lignocellulosic biomass subjected to the same treatment [47]. Table1shows that refined and commercial carrageenan were more susceptible to acid treatment, producing more HMF when compared to the untreated biomass, treated biomass, and by-product. Besides, contents of formic and levulinic acid in refined and commercial carrageenan were higher when compared with those in the by-product. This can be explained because of the presence of anhydrogalactose (AHG), known as an acid-labile molecule, which rapidly de- grades into HMF and, subsequently, into formic and levulinic acids [21,48,49].

Table2shows the chemical composition and yields of generated fractions. The first part of the table shows the chemical composition of each fraction, while the second part shows the chemical composition applying a mass balance according to the yield.

The yield of biomass treated with KOH relative to the starting material (untreated biomass) was approximately 72.4 ± 6.9 (%,w/w, dry basis). There was a loss of mass of around 27% during the pretreatment with KOH because part of the biomass was dissolved and removed with the repeated washes. This shows a difference with other studies that reported yields of 89.3% [11] and 87.3% [12]. This may be due to the fact that for this paper more washes were done when

Table 1 Content of hydroxymethylfurfural (HMF), formic, and levulinic acids in the acid hydrolysate of the fractions

Samples HMF (%) Formic

acid (%)

Levulinic acid (%)

Untreated biomass 2.5 ± 0.1â 3.2 ± 0.5âb 5.2 ± 1.3âb Treated biomass 1.2 ± 0.2^b 3.5 ± 0.3â 6.1 ± 0.9âb By-product 2.3 ± 0.1â 2.5 ± 0.2^b 4.1 ± 0.9^b Refined-carrageenan 4.3 ± 0.1^c 4.5 ± 0.1^c 8.1 ± 1.0â Commercial carrageenan 3.4 ± 0.1^d 3.9 ± 0.4âc 6.6 ± 1.3âb All reported data are the average values followed by the sample standard deviations. In the columns, the values with the same letters do not differ from each other with a significance level of 0.05 (Tukey’s test, Software GraphPad Instat). Contents present in percentage (g/100 g of material in dry basis)

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compared to the cited paper. The total yields of refined carrageenan and by-product relative to the starting material (untreated biomass) were 46.7 and 19.3% (w/w, dry basis), respectively. Additionally, there was a loss of approximately 6.4% (w/w, dry basis) during the extraction process, because it was not possible to recover some of the material that remained in the filter. The yields of refined carrageenan and by-product relative to the treated biomass were 64.6 ± 6.9 and 26.6 ± 2.6 (%,w/w, dry basis), respectively. Similar extraction yields (60 and 64.5% for refined carrageenan; 23.5 and 27.8%

for by-product) have been reported in the literature [11,12].

The main component of all fractions was galactan, with values between 32.7 and 53.6%, followed by glucan (13.0– 38.4%), ashes (8.6–18.2%), sulfate groups (8.1–13.8%), insoluble aromatics (0.13–8.3%), and proteins (0.1–2.6%). The content of lipids on untreated biomass was 5.2% (w/w, dry basis). However, lipids were not analyzed after treatment with KOH, because lipids were not detected in recent studies with fractions ofK. alvarezii[11,12].

The total carbohydrate content in K. alvarezii(untreated biomass) determined in this study was 57.2%, whereas, in the literature, mean values between 50.8 and 66.5% for the same species have been reported [11,12,17,50]. Ashes are the second major component of the samples in this study reaching 15.8% for the untreated biomass. This value is con- sistent with those reported in the literature (15.5–19.2%) [11, 12,17,50]. Sulfate groups are the third major component of the samples, reaching 12.6% of the untreated biomass. This value is different from those reported in the literature (9.4– 10.1%) for the same species [11,12].

The contents of galactan and sulfate groups of refined and commercial carrageenan were similar (considering the standard deviation). The content of glucan was 52% higher in refined carrageenan, while the content of ashes was 48%

higher in commercial carrageenan. The proteins and insoluble aromatic content were very low in both refined and commercial carrageenan.

The post-treatment with KOH caused a loss of 33.0% of galactan, 21.8% of glucan, 31.6% of ashes, 88.9% of proteins, 57.5% of insoluble aromatics, and 20.6% of sulfate groups.

The second part of Table 2 helps to understand where the components of the treated biomass migrated after the extraction. Galactan, ashes, and sulfate groups migrated mostly to the refined carrageenan fraction (83.4, 78.5, and 62.3%, respectively), while glucan and insoluble aromatics migrated mostly to the by-product fraction (76.1 and 91.7%, respectively). Consequently, the by-product showed an enrichment of glucan and insoluble aromatics. The protein content was divided almost evenly between the by-product and refined carrageenan.

Enzymatic Hydrolysis of the By-Product from the Processing of Refined Carrageenan

Table3shows the enzymatic activities and protein content of the commercial enzyme extracts used in this study. The cellulase extracts, Cellic CTec II, Cellulase, and Celluclast, exhib- i t e d a t o t a l c e l lu la s e a c t iv it y o f 8 8 . 2 , 2 8 . 4 , a n d 24.6 FPU mL⁻¹, respectively, and a low or undetected galactanase activity. Recent studies reported activities of Cellic CTec II between 92 and 114 FPU mL⁻¹[11,12,51]

and Celluclast between 76 and 92 FPU mL⁻¹[3,51,52]. In contrast, pectinase extracts (Pectinase, Smash XXL, and Ultra Clear) showed a galactanase-specific activity between 0.2 and 0.3 UI mg⁻¹and a low total cellulase-specific activity. Most methods for the determination of galactanase activity use gum Arabic as substrate [40,53,54]. However, using this substrate, galactanase activity was not detected in some enzymatic Table 2 Yield and chemical composition of fractions generated in the carrageenan process fromK. alvarezii

Samples Yield of sample

(g/100 g of algal biomass) (%)

Galactan (%) Glucan (%) Ashes (%) Proteins (%) Insoluble aromatics (%)

Sulfate groups (%) Components of samples (% on pulp basic)

Untreated biomass – 44.8 ± 3.4 12.4 ± 1.8 15.8 ± 0.1 2.2 ± 0.1 4.0 ± 1.0 12.6 ± 0.6

Treated with KOH 72.4 ± 6.9 41.5 ± 0.9 13.4 ± 1.8 14.9 ± 0.1 0.4 ± 0.1 2.4 ± 0.1 13.8 ± 0.4

By-product 26.6 ± 2.6 32.7 ± 3.1 38.4 ± 1.6 8.6 ± 0.2 0.2 ± 0.0 8.3 ± 1.0 8.1 ± 0.2

Refined carrageenan 64.6 ± 1.2 53.6 ± 2.6 12.3 ± 0.5 18.2 ± 0.1 0.05 ± 0.0 0.13 ± 0.0 13.3 ± 1.0

Commercial carrageenan – 44.3 ± 4.4 5.9 ± 0.3 34.8 ± 0.1 0.1 ± 0.01 0.7 ± 0.1 12.8 ± 0.1

Components of samples (% of original material)

Untreated biomass – 44.8 ± 3.4 12.4 ± 1.8 15.8 ± 0.1 2.6 ± 0.5 4.0 ± 1.0 12.6 ± 0.6

Treated with KOH 72.4 ± 6.9 30.0 ± 0.7 9.7 ± 1.3 10.8 ± 0.1 0.4 ± 0.2 1.7 ± 0.1 10.0 ± 0.3

By-product 26.6 ± 2.6 8.7 ± 0.8 10.2 ± 0.4 2.3 ± 0.1 0.1 ± 0.0 2.2 ± 0.3 2.2 ± 0.1

Refined carrageenan 64.6 ± 1.2 34.6 ± 1.7 7.9 ± 0.3 11.7 ± 0.1 0.1 ± 0.0 0.08 ± 0.0 8.6 ± 0.6 Lipids content in algal biomass: 5.2 ± 1.7 (%,w/w, pulp basic). Contents presented in percentage (g/100 g of basic and original material in dry basis. All reported data are the average values followed by the sample standard deviations

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preparations (data not shown). Therefore, a galactanase activity assay using carrageenan (0.1%,w/v) was performed and found to have a certain degree of galactanase activity among the pectinase extracts.

Figure2shows the kinetic profiles of the enzymatic hydrolysis of the by-product fraction with commercial extracts containing activities of cellulases and galactanases under study.

After 72 h of hydrolysis, a total conversion of glucan to glucose was achieved using any of the cellulase extracts (Cellic CTec II, Celluclast, and Cellulases). However, the Cellic CTec II extract showed a higher rate hydrolysis. In the first 4 h of biocatalysis, the conversion exceeded 60%, while using the extracts Celluclast and Cellulases, the conversion was between 30 and 40%, respectively (Fig.2a). Masarin et al. [11]

and Roldán et al. [12] also reported glucan conversion percentages of approximately 60% after 4 h of hydrolysis and 100% glucan conversion after 72 h, using Cellic CTec II.

The only cellulase extract that was able to hydrolyze the fraction of galactans contained in the by-product was Cellic CTec II, achieving a conversion of 14.7% after 72 h of hydrolysis (data not shown).

The enzymatic hydrolysis was evaluated using commercial pectinase extracts (Pectinase, Smash XXL, and Ultra Clear).

After 72 h of hydrolysis, no enzymatic extract reached 100%

of conversion of glucan (Fig. 3b). However, extract Ultra Clear showed a better conversion of glucan than Pectinases and Smash XXL, reaching a 67.3% of conversion of glucan at the end of the 72 h hydrolysis. Smash XXL showed limited efficiency in the conversion of glucan to glucose. This was expected because this extract showed a very low cellulase activity (Table 3). The glucose concentration decreased at the end of the hydrolysis using the commercial extract Pectinases (Sigma), probably due to the action of a glucose oxidase enzyme or some other compound reacting with glucose. The only pectinase extract that was able to hydrolyze the fraction of galactan contained in the by-product was Smash XXL, achieving a conversion of 6.5% after 72 h of hydrolysis (data not shown).

The enzymatic hydrolysis of the by-product was also evaluated using the commercial enzymatic extract Cellic CTec II with an enzymatic load of 100 FPU g⁻¹of by-product (10 times higher than previously used) to improve the hydrolysis of the galactan fraction. The conversion of glucan and galactan after 72 h of hydrolysis was approximately 100 and 30%, respectively (Fig. 3). With the use of this enzymatic load, it is possible to observe an almost complete hydrolysis of the glucan fraction in the first 4 h of hydrolysis due to the high activity of cellulolytic enzymes contained in the Cellic CTec II (Fig.3). Also, a significant increase in the conversion of galactan was observed (30%) when compared to the enzymatic hydrolysis using the same extract, but with an enzymatic load of 10 FPU g⁻¹of by-product (14%) (Fig.3).

Table4shows the kinetic parameters (gmax,k, andk∗gmax= maximum rate of glucose formation) and theR²values for each enzymatic extract and enzyme load used in the hydrolysis of the by-product obtained post-carrageenan process of K. alvarezii[42,44,45]. The values ofgmax,k, andR²varied between 4.14 and 9.64 g L⁻¹, 0.08 and 0.48 h⁻¹, and 0.8362 and 0.9889, respectively, while the maximum rate of glucose formation ranged from 0.71 to 4.33 g L⁻¹h⁻¹. The last param- eter was used to compare the effects of different experimental conditions on the enzymatic hydrolysis of by-product from the carrageenan process by applying a statistical test (Tukey’s test). Thus, the higher values were achieved using Cellic Table 3 Enzymatic activities and protein content of commercial

enzyme extracts Commercial extract

Total protein (mg mL⁻¹)

Total cellulases (FPU mg⁻¹)

Galactanases (UI mg⁻¹) Cellic

CTec II

68.4 1.43 0.01

Cellulase 40.0 0.91 nd

Celluclast 25.4 1.11 nd

Pectinase 22.1 0.09 0.2

Smash XXL

4.8 0.02 0.3

Ultra Clear 20.6 0.03 0.2

All reported data are the average values followed by the sample standard deviations. nd, not detected

0 2 4 6 8 10

0 20 40 60 80 100

0 20 40 60 80

Glucose (g.L-1)

Time (hours) Cellic CTec II Celluclast Cellulases

a

0 2 4 6 8 10

0 20 40 60 80 100

0 20 40 60 80

Glucose (g.L-1)

Glucan conversion (%)

Time (hours) Pectinases Smash XXL Ultra Clear Fig. 2 Glucan conversion and b

glucose concentration after enzymatic hydrolysis using (a) cellulase and (b) pectinase commercial extracts

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CTec II with an enzyme load of 100 FPU g⁻¹(4.33 g L⁻¹h⁻¹), followed by the assay using the same extract with an enzyme load of 10 FPU g⁻¹(1.72 g L⁻¹h⁻¹). There was no statistical d i f f e r e n c e b e t w e e n t h e a s s a y s u s i n g C e l l u c l a s t (0.89 g L⁻¹ h⁻¹), Cellulases (0.71 g L⁻¹h⁻¹), Ultra Clear (0.74 g L⁻¹h⁻¹), and Pectinex (0.96 g L⁻¹h⁻¹).

Mild Acid Treatment After the Enzymatic Hydrolysis of the By-Product

According to the planning, enzymatic hydrolysis experiments were performed using commercial extract Cellic CTec II in the following three different conditions: enzyme load of 100 FPU g⁻¹for 72 h; enzyme load of 100 FPU g⁻¹for 4 h;

and enzyme load of 10 FPU g⁻¹for 4 h. After that, the hydrolysates were submitted to an acid hydrolysis in mild conditions of temperature (T), time (t), and acid concentration (C) set by a 2³full factorial experimental design with three replicates at the center point. DoE (Design of Experiments) methodology was used because through it, the individual and interaction effects of the independent variables on the system response variable can be evaluated with a lower experimental effort, in contrast to the usual practice of varying a factor while the others are kept constant (one-factor-at-time methodology).

For statistical analysis of the experimental design data, it is necessary that the real values of the independent variables be coded as follows:

X_i¼ Z_i−Z_i;0

=Δh_i; i¼1;2;3 ð2Þ

In Eq. (2),Xi= coded value of the variableZi;Zi, 0= value ofZiat the center point, andΔhi=ΔZi/2= change step. Thus, the coded values of temperature (X1), time (X2), and acid concentration (X3) are given by the following equations:

X1 ¼ðT−70^oCÞ=10^oC ð3Þ

X2 ¼ðt−60 minÞ=30 min ð4Þ

X3 ¼ðC−1:25%Þ=0:75% ð5Þ

Table5shows the real and coded values of the independent variables (temperature, time, and acid concentration) and the values of the response variables.

The glucose and galactose were considered as desired products (the higher the concentration, the better the result), while the HMF was considered as an undesired product (the lower the concentration, the better the result). These consider- ations take into account fermentative process in which yeast consumes glucose and galactose as carbon source and HMF cause damage in yeast by reducing enzymatic and biological activities, breaking down DNA and inhibiting RNA and protein synthesis [22,23]. After the acid treatment, the hydrolysate did not show the gel form at 30 °C.

Aiming for a better interpretation of the experimental data, a variable of selectivity (S) was created and defined as the sum of glucose (Glu.) and galactose (Gal.) concentration divided by the hydroxymethylfurfural concentration (HMF), i.e.,S =(Glu.+ Gal.) /HMF. The lower theS, the more HMF and less glucose and galactose are formed. Conversely, the higher theS, the less HMF and more glucose and galactose are produced.

0 4 8 12 16

0 20 40 60 80 100

0 20 40 60 80

Concentration (g.L-1)

Conversion (%)

Time (hours)

Glucan conversion Galactan conversion Glucose concentration Galactose concentration

Fig. 3 Glucan and galactan conversion after enzymatic hydrolysis using Cellic CTec II 100 FPU g⁻¹of by-product

Table 4 Kinetic parameters of the enzymatic hydrolysis of by- product of carrageenan process fromK. alvareziiusing different enzymatic extracts and enzyme loads

Enzymatic extracts Enzyme load k(h⁻¹) gmax(g L⁻¹) k∗gmax(g L⁻¹h⁻¹)^* R² Cellic CTec II 100 FPU g⁻¹ 0.48 ± 0.1 8.99 ± 0.5 4.33 ± 0.6^a 0.9684 Cellic CTec II 10 FPU g⁻¹ 0.18 ± 0.0 9.64 ± 0.3 1.72 ± 0.2^b 0.9645

Celluclast 10 FPU g⁻¹ 0.09 ± 0.0 9.46 ± 0.4 0.89 ± 0.1^c 0.9889

Cellulases 10 FPU g⁻¹ 0.08 ± 0.0 8.70 ± 0.4 0.71 ± 0.1^c 0.9850

Ultra Clear 20 UI g⁻¹ 0.14 ± 0.0 5.28 ± 0.9 0.74 ± 0.0^c 0.9778

Pectinex 20 UI g⁻¹ 0.23 ± 0.0 4.14 ± 0.3 0.96 ± 0.1^c 0.8362

All reported data are the average values followed by the sample standard deviations

*In the column, the values with the same letters do not differ from each other with a significance level of 0.05 (Tukey’s test, Software GraphPad Instat)

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Table5Glucose(Glu.),galactose(Gal.),andhydroxymethylfurfural(HMF)concentrationsafterenzymatichydrolysisfollowedbyacidhydrolysisoftheby-productofcarrageenanprocessfrom K.alvarezii Assay1:100FPUg−1 —72hAssay2:100FPUg−1 —4hAssay3:10FPUg−1 —4h Exp.Temp.(°C)Time(min)H2SO4(%w/v)Glu.(gL−1 )Gal.(gL−1 )HMF(gL−1 )S(−)Glu.(gL−1 )Gal.(gL−1 )HMF(gL−1 )S(−)Glu.(gL−1 )Gal.(gL−1 )HMF(gL−1 )S(−) 160(−1)*30(−1)0.50(−1)14.451.550.0004734,004.713.100.600.0002553,932.55.300.040.0001052,615.8 280(+1)30(−1)0.50(−1)14.061.430.0009216,798.914.110.640.0012911,460.96.700.080.001355016.1 360(−1)90(+1)0.50(−1)14.041.500.0007420,897.412.910.450.0004132,643.85.600.060.0004113,811.9 480(+1)90(+1)0.50(−1)14.891.630.003884253.114.621.370.005632839.25.670.260.005321116.0 560(−1)30(−1)2.00(+1)13.101.360.005282739.412.950.390.006152170.45.780.300.0006110,027.0 680(+1)30(−1)2.00(+1)14.211.530.013901132.013.570.590.02996472.75.670.230.00696847.7 760(−1)90(+1)2.00(+1)13.731.620.012701209.613.690.560.008971588.65.880.190.000946456.3 880(+1)90(+1)2.00(+1)13.781.620.07913194.714.030.780.10446141.76.670.310.06383109.3 970(0)60(0)1.25(0)14.161.550.01797874.313.640.560.010411363.96.040.230.001205234.0 1070(0)60(0)1.25(0)13.871.550.009731583.914.240.670.006742213.36.620.080.001175723.3 1170(0)60(0)1.25(0)14.581.500.005742801.913.200.520.008221668.75.420.200.000856573.2 Beforeacidtreatment13.180.96––12.790.24––5.20nd–– S=(Glu.+Gal.)/HMF;nd,notdetected *Valuesinparenthesesrepresentthecodedvaluesoftheindependentvariables

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As expected, an increase of galactose concentration in all experiments was observed, achieving the highest value in assay 1 (1.63 g L⁻¹), an increase of 0.67 g L⁻¹compared with the concentration before the acid treatment. Furthermore, experiments of the assay 2 also showed an increase of galactose concentration, the highest being 1.37 g L⁻¹, an increase of 1.13 g L⁻¹compared with the concentration before the acid treatment. The highest galactose concentration in assay 3 was 0.31 g L⁻¹. Note that galactose was not detected before acid treatment in the third assay.

The HMF concentration was very low in all assays, ranging 0.47–79.13, 0.25–104.46, and 0.10–63.83 mg L⁻¹in the first, second, and third assay, respectively. These concentrations are much lower than 1 g L⁻¹, which has been shown by most studies to cause fermentation inhibition [23,55–59]. Neither formic nor levulinic acid was detected after the acid treatment.

Additionally, there was an increase in the concentration of glucose, reaching maximum values of 14.89, 14.62, and 6.70 g L⁻¹in the first, second, and third assay, respectively.

After 72 h of enzymatic hydrolysis using an enzyme load of 100 FPU g⁻¹, the glucan conversion into glucose was approximately 100% (Fig.3). However, after the acid treatment, the glucose concentration increased slightly (from 13.18 to 14.89 g L⁻¹), indicating that enzymes were not able to fully hydrolyze the glucan fraction. Also, it shows that there is a level of error in the determination of the chemical composition of the by-product, being that the content of glucan was determined as 38.4 ± 1.6% (Table2).

Statistical Analysis of Enzymatic Hydrolysis Followed by Mild-Acid Treatment

Initially, the response variables (final concentration of glucose, galactose, and HMF) were individually modeled by first-order mathematical models in which linear terms refer- ring to main effects and second-order interaction terms, plus a constant, were included. However, adjustments of the coefficients of such mathematical models resulted in values without statistical significance for two of the three investigated variables, except HMF concentration (data not shown). Due to these inconclusive results and to the multivariate characteristic of the system studied, it was convenient to transform the multi-response system into a single-response system, building a single variable that takes into account all the response variables that were desired to optimize in the process, making this variable more appropriate to represent the system response.

Considering that at the end of the process, it is desired to maximize the formation of monomeric sugars and minimize the formation of HMF, an appropriate response variable is the ratio between desired (monomeric sugars) and undesired (HMF) product concentrations, represented bySand already defined elsewhere. As previously highlighted, the variableS can be interpreted as the selectivity of the process since it

represents the ratio between the formed quantities of desired and undesired products.

Final glucose, galactose, and hydroxymethylfurfural concentration values as well as selectivity at the end of the integrated process are presented in Table5. The selectivity data were, then, used to fit linear mathematical models given by the following equation:

Y ¼b0þb1X1þb2X2þb3X3þb12X1X2þb13X1X3

þb23X2X3 ð6Þ

In Eq. (6),Yis the is selectivity;X1,X2, andX3are the coded independent variables in the acid treatment (temperature, time, and acid concentration, respectively);b1,b2,b3,b12,b13, and b23 are the coefficients related to the main and second-order interaction effects; andb0is the offset term, corresponding to the average of the experimental observations and to the response value at the center point predicted by the model.

The third-order interaction effect (X1* X2 *X3) was neglected because, generally, high-order interaction effects (≥3) are of questionable phenomenological significance and, also, to avoid overfitting of the mathematical model by lack of degrees of freedom.

Table6presents the estimated coefficients, their respective standard deviations, and p values for the test of statistical significance of the coefficients estimated as well as the values of the coefficient of determination (R²) and thepvalues for the lack-of-fit test of the mathematical models. According to thep value test, coefficients/models are rejected if this value is greater than the significance level (α) set for the test (usually α= 0.05) while the value ofR²provides an indication of how much of the variance in the experimental data is explained by the mathematical model.

All estimated coefficients were statistically significant at a 95% confidence level with the exception ofb12adjusted for assay 1. The main effects on the response were negative for the three investigated variables, meaning that the selectivity becomes lower when experimental conditions in the higher levels of the independent variables are used, which is higher temperature, time, and acid concentration. Regarding the interaction effects, these were favorable to the selectivity of the process, showing positive values for the three interactions. In particular, the effect of temperature–time interaction (X1*X2) can be interpreted as a factor of process severity, being the lowest of the three interaction effects calculated for all the assays.

R²of the assays was around 0.90, indicating good model fit and meaning that 90% of the variance around the mean of the experimental observations is explained by the mathematical model. Likewise, thepvalues were also favorable to the developed mathematical models and lack of fit was not detected by this statistical test. However, all responses end upBin a

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corner^ (Fig. S3), indicating that an optimum yet was not necessarily reached. In order to optimize the response variable, it is necessary to use central composite design built from the original one by the addition of experiments performed at new level combinations of the independent variables to esti- mate the curvature effect through second-order polynomial models. The experimental data of the current experimental design are not enough to adjust quadratic models. However, the first-order models presented here provide a first approximation of the response surface behavior.

FigureS3shows the selectivity response surfaces drawn with the mathematical models developed for the three performed assays (in the graphs, the value of the third missing independent variable is that of the center point). It can be observed that the highest selectivity values are verified for those experimental conditions in which the values of the con- trolled factors are minimal, as previously discussed.

Conclusion

Cellic CTec II enzymatic extract has the highest potential among those evaluated for the enzymatic hydrolysis of the polysaccharide fraction of the by-product generated in the carrageenan process fromK. alvarezii. Nevertheless, after the enzymatic hydrolysis of the by-product, there was still a gel formation in the hydrolysate. The acid treatment was efficient, breaking the gel and increasing the concentration of fermentable sugars without forming appreciable amounts of inhibitors. However, aiming at a fermentative process of the final hydrolysate (enzymatic hydrolysis followed by acid hydrolysis), it will still be necessary to

increase the consistency (mass/water ratio) in the hydrolysis steps, thus increasing glucose and galactose concentration levels in the process. Nevertheless, it will also be necessary to evaluate the effect of acid hydrolysis on HMF concentration in trials with high consistency.

However, the integrated enzymatic and acid hydrolysis of the by-product indicates that there is potential for glucose and galactose formation aiming to produce bioethanol or higher added value products, such as organic acids (butyric, acetic, succinic, and itaconic acids). In this context, the integrated hydrolysis process must be optimized for a higher production of monomeric sugars, which requires the investigation of new experimental conditions using higher order experimental designs, such as central composite designs (CCD).

Acknowledgements FAPESP, CNPq, PROPe-UNESP, and Programa de Apoio ao Desenvolvimento Científco da Faculdade de Ciências Farmacêuticas da UNESP-PADC supported this work. We appreciate, also, the support of German Enrique Pesantez (Bachelor of Science in Electrical Engineering) by providing resources for the English revision of the paper.

Availability of Supporting Data Supporting data used in the publication of this paper can be provided upon request.

Authors’Contributions EGSC and FRPC performed the chemical and enzymatic hydrolysis analyses of the samples, data interpretation, and review of the manuscript. VCG provided the experimental macroalgae strains and performed the field trials, data interpretation, and review of the manuscript. LEO, RM, SCO, and FM participated in the design of the study, data interpretation, mathematical modeling, and review of the manuscript. All authors read and approved the final manuscript.

Funding This work was supported by FAPESP (contract number 2014/05969-2), CNPq (contract number 440385/2014-8), PROPe— Table 6 Calculated coefficients

(bi) and respective standard deviations,p, andR²values for the significance test of coefficients/models

Coefficients Assay 1 Assay 2 Assay 3

b₀ 7875.2 ± 297.1

(p= 0.001420)

10,117.8 ± 124.2 (p= 0.000151)

9814.8 ± 217.0 (p= 0.000489)

b1 −4569.9 ± 348.4

(p= 0.005762)

−9541.7 ± 145.6 (p= 0.000233)

−9589.5 ± 254.5 (p= 0.000704)

b2 −3515.1 ± 348.4

(p= 0.009681)

−3954.1 ± 145.6 (p= 0.001353)

−5945.3 ± 254.5 (p= 0.001828)

b3 −8860.9 ± 348.4

(p= 0.001542)

−12,149.6 ± 145.6 (p= 0.000144)

−6973.8 ± 254.5 (p= 0.001329)

b12 132.5 ± 348.4

(p= 0.740374)

1750.4 ± 145.6 (p= 0.006848)

4830.9 ± 254.5 (p= 0.002764)

b13 3922.5 ± 348.4

(p= 0.007797)

8754.2 ± 145.6 (p= 0.000277)

5721.3 ± 254.5 (p= 0.001973)

b23 2895.8 ± 348.4

(p= 0.014167)

3722.2 ± 145.6 (p= 0.001527)

4884.4 ± 254.5 (p= 0.002704)

R² 0.874

(p= 0.012437)

0.900 (p= 0.001094)

0.909 (p= 0.005075)