Applied Science, Faculty of
Non UBC
Civil Engineering, Department of
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Sustainability 14 (24): 16383 (2022)
Dahiya, Swati
Chowdhury, Raja
Kumar, Pradeep
Ghosh, Sanjoy
Srinivasan, Asha
Algal biomass and Colocasia esculenta (Taro) leaves are available as waste biomass all over India. These biomasses can be used as renewable and sustainable resources for sugars and nutrients. Recovered nutrients and sugars can be used as cheap raw materials for biofuels and biomaterials production. The hydrolysis of dried algal biomass and Colocasia esculenta (Taro) leaves were investigated using 1%, 2%, and 5% solutions of ferric-chloride, nitric acid, and acetic acid for the reaction times of 30 and 60 min at 121 °C and 103.4 kPa (15 psi). 1% and 2% H₂SO₄ treatments were used as the reference. The solid: liquid ratio was kept at 1:10 for all the experiments. For algal biomass, a 5% acetic acid treatment for 60 min was found to be optimum with a total carbohydrate release of 44.2 mg/g biomass (solubilized monomers-0.82 mg/g of biomass) and N and P solubilization of 1.8 mg total nitrogen/g biomass and 7 mg total phosphorus/g biomass. Moreover, for Colocasia esculenta (Taro) leaves, the maximum carbohydrate yield of 95 mg/g biomass (solubilized monomers-43.6 mg/g of biomass) and nutrient solubilization of 5.02 mg total nitrogen/g biomass was obtained with 5% ferric chloride treatment for 60 min. The results obtained showed that various hydrolyzing agents used in this study acted differently on different types of biomasses. Acetic acid worked best in hydrolyzing the algal biomass, and for the hydrolysis of Taro leaves, ferric chloride and nitric acid were effective. Statistical analysis showed that the chemical concentration was one of the prime factors for releasing P from algal biomass. For carbohydrate release from Taro leaves, either time or concentration, or both, were the prime factors that affected the carbohydrate release.
Citation: Dahiya, S.; Chowdhury, R.;Kumar, P.; Ghosh, S.; Srinivasan, A.Recovery of Sugar and Nutrientsfrom Algae and Colocasia esculenta(Taro) Leaves Using ChemicalHydrolysis. Sustainability 2022, 14,16383. https://doi.org/10.3390/su142416383Academic Editor: Antonio ZuorroReceived: 4 October 2022Accepted: 27 November 2022Published: 7 December 2022Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: © 2022 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).sustainabilityArticleRecovery of Sugar and Nutrients from Algae and Colocasiaesculenta (Taro) Leaves Using Chemical HydrolysisSwati Dahiya 1 , Raja Chowdhury 1,*, Pradeep Kumar 2, Sanjoy Ghosh 3 and Asha Srinivasan 41 Department of Civil Engineering, Indian Institute of Technology Roorkee, Roorkee 247667, India2 Department of Civil and Environmental Engineering, Sharda University, Greater Noida 201310, India3 Department of Bioscience and Bioengineering, Indian Institute of Technology Roorkee, Roorkee 247667, India4 Department of Civil and Environmental Engineering, The University of British Columbia,Vancouver, BC V6T1Z4, Canada* Correspondence: rajacfce@iitr.ac.in; Tel.: +91-1332284793Abstract: Algal biomass and Colocasia esculenta (Taro) leaves are available as waste biomass allover India. These biomasses can be used as renewable and sustainable resources for sugars andnutrients. Recovered nutrients and sugars can be used as cheap raw materials for biofuels andbiomaterials production. The hydrolysis of dried algal biomass and Colocasia esculenta (Taro) leaveswere investigated using 1%, 2%, and 5% solutions of ferric-chloride, nitric acid, and acetic acid for thereaction times of 30 and 60 min at 121 ◦C and 103.4 kPa (15 psi). 1% and 2% H2SO4 treatments wereused as the reference. The solid: liquid ratio was kept at 1:10 for all the experiments. For algal biomass,a 5% acetic acid treatment for 60 min was found to be optimum with a total carbohydrate releaseof 44.2 mg/g biomass (solubilized monomers-0.82 mg/g of biomass) and N and P solubilizationof 1.8 mg total nitrogen/g biomass and 7 mg total phosphorus/g biomass. Moreover, for Colocasiaesculenta (Taro) leaves, the maximum carbohydrate yield of 95 mg/g biomass (solubilized monomers-43.6 mg/g of biomass) and nutrient solubilization of 5.02 mg total nitrogen/g biomass was obtainedwith 5% ferric chloride treatment for 60 min. The results obtained showed that various hydrolyzingagents used in this study acted differently on different types of biomasses. Acetic acid worked bestin hydrolyzing the algal biomass, and for the hydrolysis of Taro leaves, ferric chloride and nitricacid were effective. Statistical analysis showed that the chemical concentration was one of the primefactors for releasing P from algal biomass. For carbohydrate release from Taro leaves, either time orconcentration, or both, were the prime factors that affected the carbohydrate release.Keywords: chemical hydrolysis; algae; Colocasia esculenta (Taro) leaves; carbohydrate; nutrientsolubilization; acidic treatment1. IntroductionIn recent years, a surge in research on the production of bio-based products and biofu-els is observed in the scientific literature [1,2]. A portion of the petro product and fossil fueldemand is likely to be replaced by these bio-based products and biofuels produced in abiorefinery (similar to an oil refinery) [3]. The most widely used raw material is lignocel-lulosic biomass, which requires drastic pretreatment to deconstruct lignin–hemicellulosebonds to break cellulose as a simple carbohydrate.Several chemical reagents ranging from a strong acid and base, a weak acid and base,and various metallic salts (acidic or basic) were used for the pretreatment of lignocellulosicbiomass. In addition to the above-mentioned chemicals, ionic liquids, steam, and ammoniaexplosion techniques have also been used to break the lignin structure of the lignocellulosicbiomass [4–7]. Li et al. [8] investigated the effects of several metal oxides on the recovery ofsugars from corn stover. These metals worked as catalysts for monomer formation fromthe complex sugars. However, these metal oxides break the hemicellulose down into itsSustainability 2022, 14, 16383. https://doi.org/10.3390/su142416383 https://www.mdpi.com/journal/sustainabilitySustainability 2022, 14, 16383 2 of 18monomers. Recently, uses of deep eutectic solvents for pretreatment and value-addedproduct recovery from biomass have shown promising results [9].Similarly, ferric salts (i.e., ferric chloride) also help to solubilize the hemicellulosecomponent of the complex sugars. Organic acids such as acetic acid, oxalic acid, and malicacid have also been used for the solubilization of sugars from biomass. These organicacids depolymerize the cellulose into the oligomers and glucose and enhance the organicnitrogen and phosphorus release from the complex organic matter [10].The primary sources of lignocellulosic biomass are forest and agricultural residues.Nevertheless, it needs extensive pretreatment strategies to break the lignin—cellulosebonds, ultimately increasing the cost of producing simple sugars [11]. Moreover, Furfuraland 5-hydroxymethylfurfural (HMF) are degradation compounds generated during thehydrolysis of lignocellulosic biomass, which distinctly inhibit microbial growth [12]. Feed-stock contributes 70–80% of the total production cost, which can be substantially reducedby the utilization of waste and cheaper raw materials [13]. Hence, it is not possible toexploit these resources further to produce value-added products in a considerable amount.So, there is a need to look for waste biomass with a low or minimum content of lignin [14].In this regard, algae and Colocasia esculenta (Taro) leaves are two promising candidates thatcan thrive in wastewater or marshy lands.Both biomasses generated from algae and Colocasia esculenta (Taro) leaves are amenableto chemical treatment and may not require costly enzymatic hydrolysis for enhanced sugarrecovery. Hydrolysates prepared from biomass are ultimately used to produce variousvalue-added chemicals, including fuels using different microbial strains [15,16]. Variouswater bodies contain a large diversity of algal species, and some of these species can beused to produce value-added chemicals, including fuels [17]. For the production of value-added products, sugar is the main ingredient. However, nutrient contents also play a vitalrole in the production of value-added products. Chowdhury and his co-workers [18–20]showed that nutrient recovery from biomass and utilization of the same increased biofuelproduction. The produced biofuel has reduced energy demand and greenhouse gas (GHG)emissions. Hence, nutrient recovery from biomass should also be taken into account toevaluate the efficacy of a hydrolysis procedure. Optimal uses of sunlight for algae and heatproduction also reduced the cost of the algal biomass produced by wastewater treatmentplants [21].Very few studies are carried out on the hydrolysis of Colocasia esculenta (Taro) leavesand algal biomass through chemical treatment. For example, Saenphoom et al. [22] usedenzymatic treatment to hydrolyze the Taro leaves and recovered approximately 29.4 mgsugar/g of dry matter. Several researchers also explored the chemical hydrolysis of algalbiomass harvested from natural water or wastewater. Most of these hydrolysis proceduresused mineral acids. For example, Jain et al. [13] used several acids for hydrolysis proceduresand found that digestion in an autoclave with 1% H2SO4 was the most effective for releasingnutrients and sugars from algal biomass. Similarly, Castro et al. [15] investigated hydrolysisof algal biomass grown in municipal wastewater and reported that 1 M H2SO4 treatment at121 ◦C followed by enzymatic treatment released 161 g sugar/kg algal biomass. Previousstudies also explored mineral acids at different concentrations and temperatures to checktheir efficacy in releasing sugars. However, very few studies deliberated on the release ofN and P from algae and Colocasia esculenta (Taro) leaves.It is observed that a large percentage of the hydrolyzing agent ultimately ends up inthe hydrolysate. Hence, it is of great importance to use the constituents of the hydrolyzingagent as a macro or micronutrient for microbial growth. For the hydrolysis of algal biomassand Taro leaves, three chemicals were chosen. The first one was acetic acid, which showedenhanced cellulose dissolution and TKN (Total Kjeldahl Nitrogen) recovery. Acetic acid hasalso been used as a carbon source for several microorganisms that produce value-addedproducts [23–25]. Organic acids are less reactive than sulfuric acid. They do not destroyorganic materials and produce less undesirable side products than sulfuric acid [10]. Thesecond hydrolyzing reagent used was nitric acid, a vital mineral acid. Nitrate present inSustainability 2022, 14, 16383 3 of 18nitric acid can serve as a nitrogen source for microbial growth. In comparison to sulfuricacid, nitric acid causes less equipment corrosion and is more effective for hydrolyzingbiomass [26]. The third reagent explored was ferric chloride, which was found to hydrolyzehemicellulose preferentially. Iron also works as a catalyst for converting complex sugarsinto their monomers. Iron in the hydrolysate can work as a micronutrient, and some studiesshow that ferric ions positively affect lipid production by various microorganisms [27,28].FeCl3 has other unique properties such as its low cost, non-toxicity, and abundance, makingit a favorable chemical for hydrolysis [29].Therefore, the present study was designed to find the efficacy of sugar and nutrientrelease from algal bloom and Colocasia esculenta (Taro) leaves, abundantly grown in wastew-ater or marshy lands with low lignin content (algae contains no lignin). Optimum doses ofthree different chemicals, namely, ferric chloride, nitric acid, and acetic acid, were soughtusing various concentrations and time parameters to explore their effects on sugar andnutrient release.2. Materials and Methods2.1. Biomass UsedNaturally available mixed algal bloom and Colocasia esculenta (Taro) leaves werecollected from the stagnant water bodies present near the campus of the Indian Instituteof Technology Roorkee, Roorkee, India. Biomass was washed several times with distilledwater and dried in a hot-air oven at 80 ◦C for 24 h before being converted into fine powder.2.2. Hydrolyzing ReagentsAcetic acid (CH3COOH, 100% purity, ACS grade), nitric acid (HNO3, 70% purity,reagent grade), and ferric chloride anhydrous (FeCl3, 98% purity, powder) were used asthe hydrolyzing reagents. Sulfuric acid (H2SO4, 98% purity, reagent grade) was used asa reference hydrolyzing reagent for comparison. All the chemicals were purchased fromSigma Aldrich (India).2.3. Hydrolysate PreparationThe powdered forms of algae and Colocasia esculenta (Taro) leaves biomass werehydrolyzed separately using 1%, 2%, and 5% concentrations of CH3COOH, HNO3, andFeCl3 using an autoclave (at 121 ◦C and 15 psi) and two time intervals, i.e., 30 min and 1 h.The concentration of the reference hydrolyzing reagent, H2SO4 was 1%. The solid: liquidratio was kept at 1:10 for all experiments. All the hydrolysates prepared were cooled toroom temperature, before filtering, using a Whatman filter paper to remove suspendedbiomass particles. The hydrolysate was then neutralized with 0.1 M NaOH to attain apH of 7.4.2.4. Analytical Methods2.4.1. Characterization of the HydrolysatesSamples were first centrifuged at 4000 rpm for 10 min, and the supernatant was col-lected after passing through a 0.22 µm syringe filter. Chemical oxygen demand (COD)was measured using the closed reflux method (colorimetric method) given in the stan-dard methods [30]. The phenol sulphuric acid method [31] was used to estimate the totalcarbohydrates present in the hydrolysates. Absorbance was measured by a UV-VIS spec-trophotometer (DR 6000, HACH, Loveland, CO, USA). Total organic carbon (TOC) wasmeasured in a TOC analyzer (Analytik Jena, multi N/C 3100). Nitrate (NO3-), phosphate(PO4−3), and ammonia (NH4+) were measured in an Ion-chromatograph (850 professionalIC, Metrohm, Herisau, Switzerland). Metrosep A Supp 5 100/4.0 and Metrosep A Supp5 100/4.0 columns were used as anion and cation columns, respectively. Total Nitrogen(TN) was measured using the simplified TNT plus simplified TKN sTKNTM- TNT 880 kit(range:0–16 mg/L N) (HACH, USA).Sustainability 2022, 14, 16383 4 of 182.4.2. Analysis of Reducing SugarsThe estimation of simple sugars (such as glucose, xylose, etc.) and acetic acid wascarried out using high-performance liquid chromatography (HPLC). The analysis wasperformed by injecting filtered hydrolysate into HPLC (water) equipped with a Biorad®Aminex HPX-87H Column (300 × 7.8 mm) maintained at 65 ◦C. 0.005M H2SO4 was usedas a mobile phase (flow rate: 0.6 mL/min), and a refractive index detector was used for thedetection (temperature 40 ◦C).2.4.3. Statistical AnalysisAll experiments were performed in replicates. Two factors, i.e., the time of hydrolysisand the concentration of chemicals, were varied in two levels and three levels, respectively (afactorial design approach was undertaken). The obtained data were used to develop modelsto find the main effects (time and concentration) and interaction effects (time× concentration).The ANOVA and confidence interval values of each effect were estimated. R2 values of themodels generated from the experimental design were estimated. For statistical model building,a 22 factorial design was used. Hence, for the concentration, two models were produced(concentration 1 and 2% and 1 and 5%). Details about the experimental design for estimatingthe effects from the factorial design and associated statistical analysis can be found in [32].3. Results and Discussion3.1. Effect of Different Hydrolysis Procedures on Algal BiomassThe algal bloom was hydrolyzed using three different chemicals, i.e., acetic acid, nitricacid, and ferric chloride, to check its efficacy on sugar and nutrient release. Furthermore,1% H2SO4 treatment was used as the reference for the study.3.1.1. Carbohydrate and COD SolubilisationFigure 1 shows the number of total carbohydrates in the hydrolysate of algal biomassreleased by different treatments. For the acetic acid treatment, the carbohydrate released in30 min was found to be 13, 18, and 40 mg/g of biomass for 1%, 2%, and 5% concentrations,respectively. For 1 h of treatment time, the corresponding values were 18, 18, and 44.2 mg/gof biomass. Due to changes in the concentration of acetic acid from 2 to 5%, the carbohydraterelease increased more than two-fold. However, monomer production was no more than 2%of the total carbohydrate produced. Zhang et al. [33] explained that using a weak acid suchas acetic acid could help to dissolve the amorphous starch content during the hydrolysisprocess through acetylation, resulting in a decrease in the molecular packing density byreducing the formation of intermolecular hydrogen bonds. This decrease in packing densityincreases the acid permeation [23,34]. A 10% acetic acid solution was also investigated forthe hydrolysis of algal biomass at reaction times of 30 min and 1 h, releasing 15 and 29.2 mgof sugar/g of dry matter, respectively (Table S2, Supplementary Materials). Compared tothe carbohydrate release, the 5% treatment was found to be optimum for the algal biomasshydrolysis. Accordingly, hydrolysate prepared using a 10% concentration of acetic acid wasnot characterized. Models developed from the factorial design of experiments (time andconcentration are the two factors with two levels) showed that none of the effects affectedthe carbohydrate and glucose release from the algal biomass. Model 2 was a better model ifone considered the R2 value (carbohydrate release) (Table 1). Xylose released by acetic acidtreatment showed strong effects of time and concentration. Both models provided a goodfit for the experimental data.Sustainability 2022, 14, 16383 5 of 18Table 1. Statistical analysis of hydrolysate data for algae.Hydrolysing Agent (Products) Model 1 Model 2 Statistical SignificanceAcetic acid-(carbohydrate) y = 16.75 + 2.5a + 2.5b − 2.5abR2: 0.66y = 28.8 + 4.6a + 26.6b − 0.4abR2: 0.41 None of the effects were significantHNO3-(carbohydrate)y = 1.025 + 0.35a − 0.05b + 0.25abR2: 0.14y = 1.35 + 0.4a + 0.6b + 0.3abR2: 0.23 None of the effects were significantFeCl3-(carbohydrate)y = 1.15 + 0.3a + 0.1b + 0.1abR2: 0.086y = 4.45 + 0.9a + 6.7b + 0.7abR2: 0.6 None of the effects were significantAcetic acid-(glucose) y = 0.6 + 0.08a − 0.03b − 0.15abR2: 0.37y = 0.605 + 0.14a − 0.02b − 0.09abR2: 0.36 None of the effects were significantAcetic acid-(xylose) y = 0.3 + 0.1a − 0.1abR2: 0.999y = 0.25 + 0.1a − 0.1b − 0.1abR2: 0.999Model 1: effect of a and interaction significant,Model 2: all the effects were significant;Acetic acid-(PO4-P)y = 4.45 − 0.1a + 1.4b + 0.4abR2: 0.92y = 7 − 3.5a + 6.5b − 3abR2: 0.9Model 1: effect of b significant; Model 2: all theeffects were significant,HNO3-(PO4-P)y = 4.25 − 0.5a − 0.5b + 0.5abR2: 0.93y = 8.5 − 2a + 8b − abR2: 0.9Model 1: All the effects were significant,Model 2: effect of b significant;FeCl3-(PO4-P)y = 0.75 − 0.1a + 0.6bR2: 0.82y = 1.325 + 0.95a + 1.75b + 1.05abR2: 0.996Model 1: effect of b significant,Model 2: all the effects are importanta: Time, b: Concentration, 22 factorial tests were conducted. The F test was used for finding the significance of aand b (significance tests at 0.01 and 0.05 levels). Confidence intervals were also determined. R2 = Sum of square(model)/Sum of the square total. In the first column, the hydrolyzing agent used was given, and in the brackets,(), the name of the product for which the statistical model was developed was depicted. If the significance level isnot mentioned, those significance levels are at the 0.05 level.For the nitric acid treatment, the release of total carbohydrates during the 30 min ofreaction time accounted for 1.6, 0.7, and 1.3 mg/g of biomass for the 1%, 2%, and 5% con-centrations, respectively. When the treatment time was increased to 1 h, the correspondingvalues were 1.1, 1.3, and 36.3 mg/g of biomass, respectively. Glucose (29.7 mg/g of biomass)and xylose (6.6 mg/g of biomass) recovery was found in the 5% nitric acid treatment for1 h. For lower concentrations of nitric acid, the recovery of monomers was low (Figure 1).Moreover, 1% and 2% treatment conditions were not optimum in the production of glucoseand xylose. Our results correlated well with the findings of Rodriguez-Chong et al. [35], asthey pretreated Jerusalem Artichoke Stalks with HNO3 and found that 5% HNO3 (w/v)was the optimum concentration leading to the recovery of 57 mg of glucose/g of biomassat 121 ◦C after 60 min treatment. Nitric acid, a strong acid, and a powerful oxidizing agentcan produce reducing sugars by breaking the β-1,4 linkages between glucose or xylosemonomers. The treatment using nitric acid was not effective for the algal biomass becauseit does not contain hemicellulose [16,36], which is more prone to mineral acid attack. Inthis study, only 5% HNO3 treatment could release monomers, and the lesser concentrationwas not effective for hydrolysis. Statistical analysis showed that time and concentrationdid not have any significant effects on the release of carbohydrates or any of the monomers(glucose and xylose) from the algal biomass (Table 1).Similarly, for the ferric chloride treatment, only the 5% ferric chloride treatment for1 h could recover glucose (1.35 mg/g of biomass) and xylose (0.55 mg/g of biomass). Alesser concentration was not able to solubilize the carbohydrate (Figure 1). Ferric chlorideshowed interference with the phenol-sulphuric method because the color of ferric chlorideinterfered with the colorimetric estimation of carbohydrates. The solution was filteredthrough a 0.22 µm filter before the estimation of total carbohydrates. The total carbohydratecontents in the ferric chloride treated hydrolysate for 30 min were found to be 1, 1, and7 mg/g of biomass for 1%, 2%, and 5% concentrations, respectively. For 1 h, the valueswere 1.2, 1.4, and 8.6 mg/g of biomass. The lesser recovery of xylose could be due to theabsence of hemicellulose in the algal biomass. Sun et al. [37] mentioned that hydroniumions derived from ferric chloride salt were responsible for the depolymerization of hemi-cellulose by selective hydrolysis of glycosidic linkages. Hence, the hydrolysate preparedfrom algal biomass contained fewer monomers. Statistical analysis showed that time andconcentration did not have any significant effects on the release of carbohydrates or any ofthe monomers (glucose and xylose) from algal biomass (Table 1).Sustainability 2022, 14, 16383 6 of 18Sustainability 2022, 14, x FOR PEER REVIEW 6 of 19 Figure 1. Total carbohydrate and monomer concentrations (mg/g) in different hydrolysates of algae. Number in the figure shows the concentration (mg/g) of individual components (legend). Number on the top of each column shows the total carbohydrate content. Vertical bars show the error in estimation as standard deviation. Similarly, for the ferric chloride treatment, only the 5% ferric chloride treatment for 1 h could recover glucose (1.35 mg/g of biomass) and xylose (0.55 mg/g of biomass). A lesser concentration was not able to solubilize the carbohydrate (Figure 1). Ferric chloride showed interference with the phenol-sulphuric method because the color of ferric chloride interfered with the colorimetric estimation of carbohydrates. The solution was filtered through a 0.22 µm filter before the estimation of total carbohydrates. The total carbohy-drate contents in the ferric chloride treated hydrolysate for 30 min were found to be 1, 1, and 7 mg/g of biomass for 1%, 2%, and 5% concentrations, respectively. For 1 h, the values were 1.2, 1.4, and 8.6 mg/g of biomass. The lesser recovery of xylose could be due to the absence of hemicellulose in the algal biomass. Sun et al. [37] mentioned that hydronium ions derived from ferric chloride salt were responsible for the depolymerization of hemi-cellulose by selective hydrolysis of glycosidic linkages. Hence, the hydrolysate prepared from algal biomass contained fewer monomers. Statistical analysis showed that time and concentration did not have any significant effects on the release of carbohydrates or any of the monomers (glucose and xylose) from algal biomass (Table 1). For the reference study (1% H2SO4), the total carbohydrate content for the 30 min treatment was estimated as 1.7 mg/g of biomass, which included more than 50% mono-mers (glucose—0.22 mg/g of biomass, xylose—0.2 mg/g of biomass, and arabinose—0.6 mg/g of biomass). For the 1 h treatment, the recovery of total carbohydrate was 2.4 mg/g of biomass with glucose, 0.2 mg/g of biomass with xylose, and 0.2 mg/g of biomass with arabinose. Compared to the other treatments used in this study, only acetic acid could solubilize the carbohydrates in a low concentration (1% and 2%). Hence, acetic acid proved to be an effective treatment method for the hydrolysis of algal biomass. COD was estimated after subtracting COD generated from the hydrolyzing reagent present in the hydrolysate. Hence, the estimated COD was due to the sugar and other constituents generated from biomass present in the hydrolysate. A significant increase in the solubilization of COD was noted for the 5% treatment of acetic acid. However, if one assumes that all the soluble sugar is present as glucose (glucose imparts the highest COD among all the sugars), the estimated COD was higher than that imparted by sugars. Hence, it appears that there are several other soluble compounds present in the hy-drolysate, which also contributed to the COD. Lin et al. [38] also reported that many Figure 1. Total carbohydrate and monomer concentrations (mg/g) in different hydrolysates of algae.Number in the figure shows the concentration (mg/g) of individual components (legend). Numberon the top of each column shows the total carbohydrate content. Vertical bars show the error inestimation as standard deviation.For the reference study (1% H2SO4), the total carbohydrate content for the 30 mintreatment was estimated as 1.7 mg/g of biomass, which included more than 50% monomers(glucose—0.22 mg/g of biomass, xylose—0.2 mg/g of biomass, and arabinose—0.6 mg/gof biomass). For the 1 h treatment, the recovery of total carbohydrate was 2.4 mg/g ofbiomass with glucose, 0.2 mg/g of biomass with xylose, and 0.2 mg/g of biomass witharabinose. Compared to the other treatments used in this study, only acetic acid couldsolubilize the carbohydrates in a low concentration (1% and 2%). Hence, acetic acid provedto be an effective treatment method for the hydrolysis of algal biomass.COD was estimated after subtracting COD generated from the hydrolyzing reagentpresent in the hydrolysate. Hence, the estimated COD was due to the sugar and otherconstituents generated from biomass present in the hydrolysate. A significant increase inthe solubilization of COD was noted for the 5% treatment of acetic acid. However, if oneassumes that all the soluble sugar is present as glucose (glucose imparts the highest CODamong all the sugars), the estimated COD was higher than that imparted by sugars.Hence, it appears that there are several other soluble compounds present in the hy-drolysate, which also contributed to the COD. Lin et al. [38] also reported that many organiccompounds were present in the hydrolysate they prepared. TOC (Total Organic Carbon)was also estimated to deduce the prevalent oxidation state of the resultant hydrolysate.It was observed that the COD/TOC ratio of the hydrolysate prepared by the acetic acidtreatment ranged from 2–2.5 for the 5% acetic acid treatment. However, the COD/TOCratio of the most reduced form of sugar (glucose) is 2.7. Hence, it is most likely that someorganic compounds are pr sent in the hydrolys te i a less reduced form th n glucose(in glucose C has an oxidation state of 0). These organic compounds imparted the unex-plained part of the estim ted COD. The COD of o e-mole glucos is 192 g/L, wher asth TOC of one-mole glucose is 12 × 6= 72 g/L. Hence, the COD/TOC: is 2.67 (Table S1,Supplementary M t rials).In contr st to the acetic cid reatment, soluble ch mical oxygen demand (sCOD) inthe nitric-acid-treat d hydrolysate was fou d to be l wer (Figur 2). Low COD obtained inthe nitric acid treatmen was mos likely due to the fact that nitric acid is a stron cid andmight hav degraded the solubilized COD to CO2.Sustainability 2022, 14, 16383 7 of 18Sustainability 2022, 14, x FOR PEER REVIEW 7 of 19 organic compounds were present in the hydrolysate they prepared. TOC (Total Organic Carbon) was also estimated to deduce the prevalent oxidation state of the resultant hy-drolysate. It was observed that the COD/TOC ratio of the hydrolysate prepared by the acetic acid treatment ranged from 2–2.5 for the 5% acetic acid treatment. However, the COD/TOC ratio of the most reduced form of sugar (glucose) is 2.7. Hence, it is most likely that some organic compounds are present in the hydrolysate in a less reduced form than glucose (in glucose C has an oxidation state of 0). These organic compounds imparted the unexplained part of the estimated COD. The COD of one-mole glucose is 192 g/L, whereas the TOC of one-mole glucose is 12 × 6= 72 g/L. Hence, the COD/TOC: is 2.67 (Table S1, Supplementary Materials). In contrast to the acetic acid treatment, soluble chemical oxygen demand (sCOD) in the nitric-acid-treated hydrolysate was found to be lower (Figure 2). Low COD obtained in the nitric acid treatment was most likely due to the fact that nitric acid is a strong acid and might have degraded the solubilized COD to CO2. Figure 2. COD (mg/g) in different hydrolysates of algal biomass. Number in the figure shows the COD concentration (mg/g). Vertical bars show the error in estimation as standard deviation. Ferric chloride treatment was also found to be less efficient for the solubilization of COD than other hydrolyzing reagents in the case of algal biomass (Figure 2). For the 30-min treatment, the amount of COD was estimated to be 16, 15, and 27 mg/g of biomass for 1%, 2%, and 5%, respectively. For the 1 h treatment, the corresponding values were 16, 14, and 29 mg/g of biomass, respectively. Thus, the 5% treatment showed a better result than the lower concentrations. However, a change in the time of hydrolysis did not have a sig-nificant effect on the COD solubilization. Therefore, from Figures 1 and 2, it was con-cluded that only the 5% treatment is effective for the solubilization of organic matter from the algal biomass in the ferric-chloride-treated hydrolysate. For 1% H2SO4, COD solubili-zation was 9 with 17 mg/g of biomass for reaction times of 30 min and 1 h, respectively. 3.1.2. Nutrient Solubilisation (TN and TP) Nitrogen and phosphorous present in a culture medium play a significant role in the growth of microorganisms [39] as nitrogen acts as the building block for the synthesis of proteins and nucleic acid. Moreover, phosphorous is necessary for the cellular uptake of nitrate present in the media. As shown in Figures 3 and 4, the total nitrogen (TN) and orthophosphate contents were higher in all the hydrolysates compared to the reference hydrolysate (1% H2SO4 treatment). This observation indicated that the hydrolyzing Figure 2. COD (mg/g) in different hydrolysates of algal biomass. Number in the figure shows theCOD concentration (mg/g). Vertical bars show the error in estimation as standard deviation.Ferric chloride treatment was also found to be less efficient for the solubilization ofCOD than other hydrolyzing reagents in the case of algal biomass (Figure 2). For the 30-mintreatment, the amount of COD was estimated to be 16, 15, and 27 mg/g of biomass for 1%,2%, and 5%, respectively. For the 1 h treatment, the corresponding values were 16, 14, and29 mg/g of biomass, respectively. Thus, the 5% treatment showed a better result than thelower concentrations. However, a change in the time of hydrolysis did not have a significanteffect on the COD solubilization. Therefore, from Figures 1 and 2, it was concluded thatonly the 5% treatment is effective for the solubilization of organic matter from the algalbiomass in the ferric-chloride-treated hydrolysate. For 1% H2SO4, COD solubilization was9 with 17 mg/g of biomass for reaction times of 30 min and 1 h, respectively.ificleic acid. Moreover, phosphorous is n cessary for the cellular uptakeof nitrate prese t in the media. As shown in Figures 3 and 4, the total nitrogen (TN)and ortho osphate contents were higher in all the hydrolysates compared to th refer-ence h drolysate (1% H2SO4 treatment). This observation indicated that the r l zireagents used in the present study were able to elevate the extraction of nutrients from thealgal biomass.The total nitrogen in this study (Figure 3) comprises Nitrate-N and TKN-N. TKN(TKN is used to express TKN-N from this point onward) was estimated only for samplesthat showed enhanced sugar recoveries. The nitrate-N content for the acetic acid-treatedhydrolysate was very low, i.e., between 0.2 and 0.3 mg/g of biomass, and TKN wasestimated to be approximately 2 mg/g of biomass. For nitric acid, the value of nitratewas found to be higher in all hydrolysates because of the presence of nitrogen (as nitrate)in the hydrolyzing reagent itself. Therefore, TKN analysis was not performed for thenitric-acid-treated hydrolysates.Sustainability 2022, 14, 16383 8 of 18Sustainability 2022, 14, x FOR PEER REVIEW 8 of 19 reagents used in the present study were able to elevate the extraction of nutrients from the algal biomass. The total nitrogen in this study (Figure 3) comprises Nitrate-N and TKN-N. TKN (TKN is used to express TKN-N from this point onward) was estimated only for samples that showed enhanced sugar recoveries. The nitrate-N content for the acetic acid-treated hydrolysate was very low, i.e., between 0.2 and 0.3 mg/g of biomass, and TKN was esti-mated to be approximately 2 mg/g of biomass. For nitric acid, the value of nitrate was found to be higher in all hydrolysates because of the presence of nitrogen (as nitrate) in the hydrolyzing reagent itself. Therefore, TKN analysis was not performed for the nitric-acid-treated hydrolysates. Figure 3. Total nitrogen and nitrate-N (mg/g) present in different hydrolysates of algal biomass. Number in the figure shows the concentration (mg/g) of individual components (lagend). Vertical bars show the error in estimation as standard deviation. For the ferric chloride treatment, the nitrate content was between 0.2 and 0.6 mg/g of biomass in all the hydrolysates for 30 min and 1 h of reaction time. TKN was estimated to be 1.2 mg/g of biomass for the 5% treatment (1 h). The nitrate values were lower in this case as compared to the nitric acid treatment. As compared to the 1% H2SO4 treatment (reference hydrolyzing agent), which resulted in nitrate recovery of 0.2 mg/g of biomass for both the 30 min and 1 h treatments, the hydrolysate produced using acetic acid had a higher amount of nitrate content. The concentration of hydrolyzing reagents and reaction time did not have a significant effect on the TN recovery. PO4−3-P content (Figure 4) for acetic-acid-treated hydrolysates at 1%, 2%, and 5% con-centrations was 4, 5, and 13.5 mg/g of biomass, respectively, for the 30 min reaction time. For the 1 h treatment, the corresponding values were 3.5, 5.3, and 7 mg/g of biomass. A higher amount of phosphorus was found in the 5%-treated hydrolysate, and it was re-duced when the treatment time was increased from 30 min to 1 h. Statistical analysis also revealed that for model 1, concentration had a significant influence on phosphorus solu-bilization. Model 2 depicted that all effects were significant for phosphorus solubilization. However, time and interaction terms had a negative effect on P solubilization (Table 1). Figure 3. Total nitrogen and nitrate-N (mg/g) present in different hydrolysates of algal biomass.Number in the figure shows the concentration (mg/g) of individual components (lagend). Verticalbars show the error in estimation as standard deviation.For the ferric chloride treatment, the nitrate content was between 0.2 and 0.6 mg/g ofbiomass in all the hydrolysates for 30 min and 1 h of reaction time. TKN was estimated tobe 1.2 mg/g of biomass for the 5% treatment (1 h). The nitrate values were lower in thiscase as compared to the nitric acid treatment. As compared to the 1% H2SO4 treatment(reference hydrolyzing agent), which resulted in nitrate recovery of 0.2 mg/g of biomassfor both the 30 min and 1 h treatments, the hydrolysate produced using acetic acid had ahigher amount of nitrate content. The concentration of hydrolyzing reagents and reactiontime did not have a significant effect on the TN recovery.PO4−3-P content (Figure 4) for acetic-acid-treated hydrolysates at 1%, 2%, and 5%concentrations was 4, 5, and 13.5 mg/g of biomass, respectively, for the 30 min reaction time.For the 1 h treatment, the corresponding values were 3.5, 5.3, and 7 mg/g of biomass. Ahigher amount of phosphorus was found in the 5%-treated hydrolysate, and it was reducedwhen the treatment time was increased from 30 min to 1 h. Statistical analysis also revealedthat for model 1, concentration had a significant influence on phosphorus solubilization.Model 2 depicted that all effects were significant for phosphorus solubilization. However,time and interaction terms had a negative effect on P solubilization (Table 1).In the case of nitric acid, the phosphate-p values (Figure 4) were found to be higherfor the 5% concentration with 14 and 11 mg/g of biomass for 30 min and 1 h treatment,respectively. The pattern of the results was similar to the acetic-acid-treated hydrolysates asthe maximum amount of phosphorus was obtained in the 5% nitric-acid-treated hydrolysate.Statistical analysis also revealed that concentration was the main parameter that affectedthe P solubilization (Table 1).Ferric chloride was the least efficient chemical for the recovery of phosphorus from thealgal biomass. For ferric chloride treatment, neither the concentration nor time positivelyaffected phosphorus recovery from the biomass. Estimated P in the ferric-chloride-treatedhydrolysates was 0.5, 1.1, and 1.2 mg/g of biomass for 1%, 2%, and 5%, respectively, for the30 min reaction. For the 1 h reaction, the corresponding values were 0.4, 1, and 3.2 mg/g ofbiomass. Statistical analysis showed that concentration and other effects were importantdepending on the model. Model 2 provided a better estimate of variance (R2) as comparedto model 1 (Table 1).Sustainability 2022, 14, 16383 9 of 18Sustainability 2022, 14, x FOR PEER REVIEW 9 of 19 Figure 4. Phosphate-P content (mg/g) in hydrolysates prepared from algal biomass. Numbers in the figure show the PO4-P concentration (mg/g). Vertical bars show the error in estimation as standard deviation. In the case of nitric acid, the phosphate-P values (Figure 4) were found to be higher for the 5% concentration with 14 and 11 mg/g of biomass for 30 min and 1 h treatment, respectively. The pattern of the results was similar to the acetic-acid-treated hydrolysates as the maximum amount of phosphorus was obtained in the 5% nitric-acid-treated hy-drolysate. Statistical analysis also revealed that concentration was the main parameter that affected the P solubilization (Table 1). Ferric chloride was the least efficient chemical for the recovery of phosphorus from the algal biomass. For ferric chloride treatment, neither the concentration nor time posi-tively affected phosphorus recovery from the biomass. Estimated P in the ferric-chloride-treated hydrolysates was 0.5, 1.1, and 1.2 mg/g of biomass for 1%, 2%, and 5%, respec-tively, for the 30 min reaction. For the 1 h reaction, the corresponding values were 0.4, 1, and 3.2 mg/g of biomass. Statistical analysis showed that concentration and other effects were important depending on the model. Model 2 provided a better estimate of variance (R2) as compared to model 1 (Table 1). For the reference study (1% H2SO4), the orthophosphate content was 6.4 mg/g of bi-omass for both the 30 min and 1 h treatments. Solubilization of phosphorus bound in biomass was higher in the 5% acetic-acid- and nitric-acid-treated hydrolysates than in the H2SO4-treated hydrolysate. Ferric chloride was inefficient for the recovery of phosphorus even in the higher concentration. The low con-centration of phosphate found in the hydrolysate could be due to the formation of insol-uble iron phosphate [40]. For P solubilization, concentration was the most critical param-eter. However, in some statistical models, concentration and time were the significant pa-rameters for P release (Table 1). 3.2. Comparison of Hydrolysates Prepared from Algal Biomass Several researchers have tried to hydrolyze the algal biomass through various chem-ical treatments. A comparative study of sugar release was carried out to evaluate the at-tractiveness of the hydrolyzing reagents used in the present study compared to other chemicals reported in the literature (Table 2). In the present study, 44 mg of carbohy-drate/g biomass was obtained for acetic acid treatment, and COD solubilization was also high. However, as these studies did not provide the recovery of other nutrients, a decision Figure 4. Phosphate-P content (mg/g) in hydrolysates prepared from algal biomass. Numbersin the figure show the PO4-P concentration (mg/g). Vertical bars show the error in estimation asstandard deviation.For the reference study (1% H2SO4), the orthophosphate content was 6.4 mg/g ofbiomass for both the 30 min and 1 h treatments.Solubilization of phosphorus bound in bio ass was higher in the 5% acetic-acid- andnitric-acid-treated hydrolysates than in the H2SO4-treated hydrolysate. Ferric chloridewas inefficient for the recovery of phosphorus even in the higher concentration. The lowconcentration of phosphate found in the hydrolysate could be due to the formation ofinsoluble iron phosphate [40]. For P solubilization, concentration was the most criticalparameter. However, in some statistical models, concentration and time were the significantparameters for P release (Table 1).3.2. Comparison of Hydrolysates Prepared from Algal BiomassSeveral researchers have tried to hydrolyze the algal biomass through vario s chemicaltreatments. A comparative study of sugar release was carried out to evaluate the attractive-ness of the hydrolyzing reagents used in the present study compared to other chemicalsreported in the literature (Table 2). In the present study, 44 mg of carbohydrate/g biomasswas obtained for acetic acid treatment, and COD solubilization was also high. However, asthese studies did not provide the recovery of other nutrients, a decision to select the bestchemical for hydrolysis could not be deduced. In this study, the 5% nitric acid treatmentresulted in the highest TN and TP c ntent; however, the carbohydrate solubilization anthe soluble CODs were low, which were ssential for using a hydrolysate as a growthmedium for microorganisms. A low concentratio (1% and 2%) f acetic acid treatmentcould hydrolyze the algal biomass and release more carbohydrates and COD than otherchemical treatments. Among the hydrolysates, acetic acid proved to be the best treatmentfor the hydrolysis of algal biomass. The optimal treatment condition was the 5% acetic acidtreatment for 60 min.Sustainability 2022, 14, 16383 10 of 18Table 2. Comparison of sugar recovery from algal biomass using various chemical treatmentsreported in the literature vs. present study.Reference Treatment ResultWu et al. [41] 5% H2SO4 51.6 mg/g of glucoseCastro et al. [15] 10% H2SO4sugar yield was 161.1 mg/g ofbiomassJain et al. (2017) [13] 1% H2SO442.4 mg COD/g,10.6 mg TOC/g,0.086 mg TP/g39 mg TN/gDziekonska-Kubczak et al. (2018) [26] Treated Jerusalem Artichoke Stalks with5% HNO3 (w/v) at 121 ◦C in 60 min.57 mg of glucose/g of biomassOur study 5% HNO35% Acetic acid36.3 mg/g40–44 mg/3.3. Characteristics of Hydrolysate Prepared from Taro LeavesThe hydrolysates prepared via the chemical treatment of Taro leaves are given below.3.3.1. Carbohydrate and COD Solubilization for Taro LeavesThe cell wall of Taro leaves is different from algal biomass as it mainly containscellulose (1,4-β D-glucans) and non-cellulosic polysaccharides (galacturonorhamnans,galactomannan, glucomannan, and arabinoxylan) with β-1,4 linkages [42]. It was observedfrom this study that the same chemicals acted differently for different biomass. Figure 5shows the carbohydrate and monomer content in the hydrolysate of Colocasia esculenta(Taro) leaves. Acetic acid has been reported to decrease cellulose crystallinity by altering thecellulose structure with high hydrogen bond basicity (>1.0). Pin et al. [43] summarized thatacetate anions can permeate into lignin and break the O-4 bonds between carbohydrates(cellulose and hemicelluloses) and lignin to improve the overall hydrolysis efficiency. Inthe present study, monomers (mg/g biomass) in the 1% acetic-acid-treated hydrolysatewere 15.4 for glucose and 21.8 for xylose for the 30 min treatment, and 6.58 for glucose and8.58 for xylose for the 1 h treatment. By increasing the concentration of acetic acid to 2%,the percentage of monomers was almost 100% of the recovered carbohydrate. For the 5%acetic acid treatment, sugars (mg/g biomass) were found to be low, i.e., 7.93 for glucoseand 10.12 for xylose for the 30 min treatment and 7.17 for glucose and 8.05 for xylose forthe 1 h treatment. The amount of reduced sugar started decreasing with an increase inthe concentration of chemicals, which indicated that increasing the chemical concentrationled to the oxidation of the monomers in the resultant hydrolysate. Statistical analysis ofcarbohydrate data showed that the concentration and all the effects were significant forhydrolysis prepared by acetic acid. For monomers, in most of the models, all the effectswere also statistically significant. R2 values were also more than 0.9 for most models(Table 3).Results obtained in the present study were similar to those reported by [31], in whichrice straw was treated with 4.5% of acetic acid for 1 h and the recovery of sugars was asfollows: Glucose—4.6 mg/g, xylose—5.6 mg/g, and arabinose—13.4 mg/g. As comparedto the other chemical treatments (Figure 5), being a weak acid, acetic acid could not break thelignin–carbohydrate bonds to facilitate carbohydrate solubilization and was not effectivefor the hydrolysis of Colocasia esculenta (Taro) leaves.As compared to cellulose, hemicelluloses are made of C5 (xylose and arabinose) andC6 sugars (mannose, glucose, and galactose) [22]. Hemicellulose has an amorphous andheterogeneous structure. Such a structure makes it susceptible to hydrolysis [44]. Duringthe initial phase of acid pretreatment, oligomers of various chain lengths were produced dueto acid-mediated hydrolysis. Later, these oligomers of varying degrees of polymerizationcontinued to break down into monomers [45]. In the present study, the reduced sugarsfor nitric acid treatment were the highest at a 5% concentration (Figure 5). Sun et al. [37]also used nitric acid (6%) to treat sugarcane bagasse and recovered 18.6 mg of xylose/g,20.4 mg of arabinose/g, and 28.7 mg of glucose/g. If one compared the results reported bySun et al. [37], the recovery of arabinose was lower in our study but glucose and xyloseSustainability 2022, 14, 16383 11 of 18recovery were almost similar. Statistical analysis of carbohydrates released during HNO3treatment indicated that depending on the treatment, either time or concentration, or both,were significant parameters that affected the carbohydrate or monomer’s release. Thesemodels also showed that, most often, these significant parameters provided positive effectson carbohydrate release (Table 3).Sustainability 2022, 14, x FOR PEER REVIEW 11 of 19 Figure 5. Total carbohydrate and its monomers (mg/g) in the hydrolysates of Colocasia esculenta (Taro) leaves. Numbers in the figure show the concentration (mg/g) of individual components (la-gend). Numbers on the top of each column show the total carbohydrate content. Vertical bars show the error in estimation as standard deviation. Table 3. Statistical analysis of hydrolysate data for Taro leaves. Hydrolysing Agent (Products) Model 1 Model 2 Statistical Significance Acetic acid-(carbohy-drate) y = 30.05 − 3.1a + 0.9b + 12.1ab R2: 0.96 y = 43.3 − 10.6a + 27.4b + 4.6ab R2: 0.99 Model 1: interaction effect signif-icant, Model 2: main effects and inter-action are significant, In model 2 confidence interval is significant for concentration HNO3-(carbohydrate) y = 19.78 + 21.26a + 1.84b + 11.06ab R2: 0.89 y = 30.875 + 7.15a + 20.35b − 3.05ab R2:0.82 Model 1: Effect of a was signifi-cant, Model 2: b significant; FeCl3-(carbohydrate) y = 74.75 + 10a + 39.5b − 4ab R2: 0.94 y = 76.5 + 5a + 43b − 9ab R2: 0.91 b significant for both the models, H2SO4-(carbohydrate) y = 88.5 + 25a + 9b + 49ab R2: 0.9 none Interaction effect significant (F 0.01) Acetic acid –(glucose) y = 9.8075 − 5.835a − 2.365b + 2.985ab R2: 0.96 y = 9.2775 − 4.775a − 3.425b + 4.045ab R2: 0.95 All the effects were significant (model 1 and model 2) HNO3-(glucose) y = 13.765 + 18.09a + 10.19b + 14.23ab R2: 0.9 y = 15.16 + 3.48a + 12.98b − 0.38ab R2: 0.96 Model 1: a and interaction effects were significant, Model 2: b significant, FeCl3-(glucose) y = 8.22 − 1.01a + 3.23b + 0.58ab R2: 0.56 y = 14.077 + 1.755a + 14.945b + 3.345ab R2: 0.91 Model 1: none of the effects was significant, Model 2: b was significant, H2SO4-(glucose) y = 19.885 + 0.77a − 2.54b + 0.26ab R2: 0.234 none None of the effects was signifi-cant Figure 5. Total carbohydrate and its monomers (mg/g) in the hydrolysates of Colocasia esculenta (Taro)leaves. Numbers in the figure show the concentration (mg/g) of individual components (lagend).Numbers on the top of each column show the total carbohydrate content. Vertical bars show the errorin estimation as standard deviation.As these biomass feedstocks have cellulose and its breakdown produces monomers,the acid hydrolysis of pure cellulose using the above chemicals was explored. The main goalwas to examine the hydrolysis of cellulose into its monomers, and the results obtained werecompared with the hydrolysis of biomass (algae and Taro leaves) to determine the fficie cyof the reagents used in this study. The glucose content obtained by the hydrolysis of biomass(Figure 5) was higher than the pure cellulose powder (Figure S1), which indicated thathemicellulose (present in the Taro leaves) solubilization led to the formation of a glucosemonomer along with xylose. Hence, the glucose content observed in Figure 5 was due tothe dissolution of both cellulose and hemicellulose. Acetic acid was not effective in thesolubilization of pure cellulose (Figure S1), so the monomers obtained in Figure 5 must bedue to the solubilization of hemicellulose present in the biomass of Taro leaves.Metal salt FeCl3 was reported to enhance hemicellulose solubilization with low sugardegradation and less inhibitory compound formation [46,47]. In the present study, thecarbohydrate release was the highest in the ferric-chloride-treated hydrolysate (Figure 5).The ferric chloride’s strength directly affected the hydrolysis rate as the sugar solubilizationincreased with an increase in the ferric chloride concentration. The best treatment conditionfor the solubilization of carbohydrates was 5% ferric chloride treatment for 60 mi .Sustainability 2022, 14, 16383 12 of 18Table 3. Statistical analysis of hydrolysate data for Taro leaves.Hydrolysing Agent (Products) Model 1 Model 2 Statistical SignificanceAcetic acid-(carbohydrate) y = 30.05 − 3.1a + 0.9b + 12.1abR2: 0.96y = 43.3 − 10.6a + 27.4b + 4.6abR2: 0.99Model 1: interaction effect significant,Model 2: main effects and interaction aresignificant,In model 2 confidence interval is significantfor concentrationHNO3-(carbohydrate)y = 19.78 + 21.26a + 1.84b + 11.06abR2: 0.89y = 30.875 + 7.15a + 20.35b − 3.05abR2:0.82Model 1: Effect of a was significant,Model 2: b significant;FeCl3-(carbohydrate)y = 74.75 + 10a + 39.5b − 4abR2: 0.94y = 76.5 + 5a + 43b − 9abR2: 0.91 b significant for both the models,H2SO4-(carbohydrate)y = 88.5 + 25a + 9b + 49abR2: 0.9 noneInteraction effect significant(F 0.01)Acetic acid –(glucose) y = 9.8075 − 5.835a − 2.365b + 2.985abR2: 0.96y = 9.2775 − 4.775a − 3.425b + 4.045abR2: 0.95All the effects were significant (model 1 andmodel 2)HNO3-(glucose)y = 13.765 + 18.09a + 10.19b + 14.23abR2: 0.9y = 15.16 + 3.48a + 12.98b − 0.38abR2: 0.96Model 1: a and interaction effects weresignificant,Model 2: b significant,FeCl3-(glucose)y = 8.22 − 1.01a + 3.23b + 0.58abR2: 0.56y = 14.077 + 1.755a + 14.945b + 3.345abR2: 0.91Model 1: none of the effects was significant,Model 2: b was significant,H2SO4-(glucose)y = 19.885 + 0.77a − 2.54b + 0.26abR2: 0.234 none None of the effects was significantAcetic acid-(xylose) y = 13.93 − 8.335a − 2.515b + 4.885abR2: 0.96;y = 12.14 − 7.645a − 6.105b + 5.575abR2: 0.95Model 1: a and interaction effects weresignificant,Model 2: all effects were significant,HNO3-(xylose)y = 10.855 + 11.49a − 2.39b + 5.19abR2: 0.99;y = 14.75 + 3.4a + 5.4b − 2.9abR2: 0.95Model 1: a and ab effects were significant;Model 2: all the effects were significant,FeCl3-(xylose)y = 9.2475 − 3.545a + 5.045b − 1.755abR2: 0.91y = 11.2275 + 1.535a + 9.005b + 3.325abR2: 0.92Model 1: main effects were significant,Model 2: effect of b was significant,H2SO4-(xylose)y = 17.14 + 1.24a − 4.88b + 0.76abR2: 0.42 none None of the effects was significant;Acetic acid-(PO4-P)y = 0.8 − 0.1a − 0.15b − 0.11ab;R2: 0.53y = 1.07 + 0.01a + 0.39b;R2: 0.17 None of the effects was significant;HNO3-(PO4-P)y = 0.1325 + 0.145a − 0.045b − 0.015abR2: 0.76 none Effects of a was significant,FeCl3-(PO4-P)y = 0.5625 − 0.495a − 0.065b + 0.115abR2: 0.82,y = 0.545 − 0.35a − 0.1b + 0.26abR2: 0.88Model 1: effects of a was significant;Model 2: a and interaction term weresignificant,H2SO4-(PO4-P)y = 0.3925 + 0.155a + 0.295b + 0.145ab;R2: 0.67 none None of the effects was significant;a: Time, b: Concentration, 22 factorial tests were conducted. The F test was tested for finding the significance ofa and b (significance tests at 0.01 and 0.05 levels). R2 = Sum of square (model)/Sum of the square total. In thefirst column, the hydrolyzing agent used was given and, in the brackets (), the name of the product for which thestatistical model was developed was depicted. If the significance level is not mentioned, those significance levelsare at the 0.05 level.The reaction mechanism of FeCl3 in biomass degradation is related to its Lewis acidcharacter and is mainly involved in hemicellulose solubilization [48]. According to thedata obtained from hemicellulose hydrolysis, it is hypothesized that hemicellulose wascategorized into two fragments: Fast-reacting xylan and slow-reacting xylan [49]. Thishypothesis was built on the fact that the hydrolysis rate decreased dramatically afterconversion reached approximately 70%. It is observed that xylan located in the cell wall iseasily accessible by the hydrolyzing reagent. This hemicellulose is fast-reacting, while theremaining xylan is located at a greater depth and is firmly retained within cellulose chains.The other part of the slow-reacting xylan is embedded within or attached to the lignin bylignin–carbohydrate bonds [50].In addition to the hydrolysis of hemicellulose, diluted acid pretreatment decreases thedegree of polymerization of cellulose by improving its susceptibility to enzymatic degra-dation. Pretreatment with ferric chloride has been successfully used in earlier studies fordifferent lignocellulosic biomass such as sugarcane bagasse [29,46], olive tree biomass [51],corn stover [52], and barley straw [53]. In total, 300–350 mg of sugar/g of biomass wasreleased when FeCl3-pretreated biomass was further hydrolyzed using an enzyme.In the present study, COD solubilization (Figure 6) for the ferric-chloride-treatedhydrolysate was 75, 106.3, and 135.3 mg/g of biomass within a 30 min reaction. For the1 h treatment, the corresponding values were 87.8, 100.3, and 119.7 mg/g of biomass,respectively. There is a significant increase in the COD solubilization with an increase in theferric chloride concentration. However, in the acetic acid-treated hydrolysate, there was noSustainability 2022, 14, 16383 13 of 18significant increase in the solubilization of the COD with an increase in the concentrationof the chemical or time parameters.Sustainability 2022, 14, x FOR PEER REVIEW 14 of 19 Figure 6. COD (mg/g) in the different hydrolysates of Colocasia esculenta (Taro) leaves. Numbers in the figure show the COD concentration (mg/g). Vertical bars show the error in estimation as stand-ard deviation. In the nitric-acid-treated hydrolysate, COD was very high, which showed that nitric acid was very effective in the dissolution of COD compared to other chemicals such as acetic acid and ferric chloride (Figure 6). Higher soluble COD illustrates higher solubili-zation of organic matter [54]. For the reference study, the soluble COD (Figure 6) did not show any significant change with an increase in the reaction time. Results of the present study correlated well with Pin et al. [43], who examined the effect of different pretreat-ment times (between 1 h and 3 h) on the hydrolysis of water hyacinth and concluded that there were no significant differences in the yield due to the changes in the duration of pretreatment. According to the results obtained, it was observed that contrary to the algal biomass treatment, nitric acid and ferric chloride proved to be the better hydrolyzing reagents for Colocasia esculenta (Taro) leaves, and acetic acid treatment was found to be the least effec-tive. Several studies have been carried out on the hydrolysis of Taro leaves mostly using the enzymatic treatment. However, in the present study, chemical hydrolysis was con-ducted (Table 4). The sugar yield in this study was higher than those reported in the liter-ature. Hence, for better carbohydrate release, chemicals used in the present study showed better carbohydrate recovery, especially with 5% HNO3 and 5% FeCl3, for which a release of 43.6 and 42.7 mg of sugar/g of dry matter was obtained, respectively. From the results, it is evident that the Taro leaf is one of the attractive resources for the recovery of sugars and nutrients. Table 4. Comparison of sugar recovery from Colocasia esculenta (Taro) leaves by various chemical treatments reported in literature vs. present study. Reference Treatment Reagent Reducing Sugar Yield Saenphoom et al. [22] Enzyme treatment {Hemicellulase} (1% w/v) at 60 °C for 24 h leaves of Taro leaves were treated 29.4 mg/g Yan et al. [55] Enzymatic treatment {Cellulase} (0.04 g/g) at 45 °C for 48 h, Roots of Taro plants were treated 165 mg/g Our study 5% HNO3 5% FeCl3 (leaves of Taro were treated) 43.6 mg/g 42.7 mg/g Figure 6. COD (mg/g) in the different hydrolysates of Colocasia esculenta (Taro) leaves. Numbersin the figure show the COD concentration (mg/g). Vertical bars show the error in estimation asstandard deviation.In the nit ic-acid-treated hydrolysate, CO was very high, which showed th t nitricacid was very effective in the dissol tion of COD compared to other chemicals such as aceticacid and ferric chlorid (Figure 6). High r sol ble COD illustrates high r solubilizationof organic matter [54]. For the reference study, the soluble COD (Figure 6) did not showany significant change with an increase in the reaction time. Results of the present studycorrelated well with Pin et al. [43], who examined the effect of different pretreatment times(between 1 h and 3 h) on the hydrolysis of water hyacinth and concluded that there wereno significant differences in the yield due to the changes in the duration of pretreatment.According to the results obtained, it was observed that contrary to the algal biomasstreatment, nitric acid and ferric chloride proved to be the better hydrolyzing reagents forColocasia esculenta (Taro) leaves, and acetic acid treatment was found to be the least effective.Sev ral studies have be n car ie ydrolysis of Taro leaves mostly using theenzymatic treatment. However, in the present s udy, chemical hydrolysis was ducted(Table 4). The sugar yi ld in this study was higher t an those rep rted in the literatur . Hence,for better carbohydrate release, chemicals used in the present study showed better carbohydraterecovery, especially with 5% HNO3 and 5% FeCl3, for which a release of 43.6 and 42.7 mg ofsugar/g of dry matter was obtained, respectively. From the results, it is evident that the Taroleaf is one of the attractive resources for the recovery of sugars and nutrients.Table 4. Comparison of sugar recovery from Colocasia esculenta (Taro) leaves by various chemicaltreatments reported in literat resent study.Reference Treatment Reagent Reducing Sugar YieldSaenphoom et al. [22]Enzyme treatment {Hemicellulase}(1% w/v) at 60 ◦C for 24 h leaves ofTaro leaves were treated29.4 mg/gYan et al.[55]Enzymatic treatment {Cellulase}(0.04 g/g) at 45 ◦C for 48 h, Roots ofTaro plants were treated165 mg/gOur study 5% HNO35% FeCl3 (leaves of Taro were treated)43.6 mg/g42.7 mg/gSustainability 2022, 14, 16383 14 of 183.3.2. Nutrient Solubilisation (TN and TP)Nutrient recovery from biomass would reintroduce these nutrients as fertilizers. Suchrecovery also helps to reduce the load on the limited phosphorus reserve and also helps toreduce the unintended release of these nutrients in water and soil [40]. Figure 7 shows thetotal nitrogen and nitrate (mg/g) present in the hydrolysates of Taro leaves prepared usingdifferent treatments. TKN was performed only for those samples, which showed enhancedsugar recoveries. Figure 8 shows the orthophosphate content present in the hydrolysates ofTaro leaves.Sustainability 2022, 14, x FOR PEER REVIEW 15 of 19 3.3.2. Nutrient Solubilisation (TN and TP) Nutrient recovery from biomass would reintroduce these nutrients as fertilizers. Such recovery also helps to reduce the load on the limited phosphorus reserve and also helps to reduce the unintended release of these nutrients in water and soil [40]. Figure 7 shows the total nitrogen and nitrate (mg/g) present in the hydrolysates of Taro leaves pre-pared using different treatments. TKN was performed only for those samples, which showed enhanced sugar recoveries. Figure 8 shows the orthophosphate content present in the hydrolysates of Taro leaves. Figure 7. Total nitrogen and nitrate (mg/g) in different hydrolysates of Colocasia esculenta (Taro) leaves. Numbers in the figure show the concentration (mg/g) of individual components (lagend). Vertical bars show the error in estimation as standard deviation. Figure 8. Phosphate-P (mg/g) in different hydrolysates of Colocasia esculenta (Taro) leaves. Number in the figure shows the PO4-P concentration (mg/g). Vertical bars show the error in estimation as standard deviation. For acetic acid treatment, nitrate-N (Figure 7) was found to be 0.01–0.02 mg/g of bio-mass for both 30 min and 1 h reaction times. TKN was estimated to be 2.05 mg/g of bio-mass for the 5% treatment for 30 min. There was an insignificant change in the nitrate Figure 7. Total nitrogen and nitrate ( g/g) in different hydrolysates of Colocasia esculenta (Taro)l . i fi t conce tr ti ( ) i i i l t l ).ertical bars sho the error in esti ation as standard deviation.Sustainability 2022, 14, x FOR PEER REVIEW 15 of 19 3.3.2. Nutrient Solubilisation (TN and TP) Nutrient recovery from biomass would reintr duce these nutrients as fertilizers. Such recovery also helps to reduce the load on the limit d phosphorus reserve and also helps to reduce the uninte ded releas of these nutrients i water and soil [40]. Figure 7 shows the total nitrogen a d itrate (mg/g) present i he hydrolysates of Taro leaves pre-pared using dif ere t t . TKN was performed only for those samples, which showed enhanced sugar recoveries. Figure 8 s ows the orthophosphate conte t present in the hydrolys tes of Taro leaves. Figure 7. Total nitrogen and nitrate (mg/g) in different hydrolysates of Colocasia esculenta (Taro) leaves. Numbers in the figure show the concentration (mg/g) of individual components (lagend). Vertical bars show the error in estimation as standard deviation. Figure 8. Phosphate-P (mg/g) in different hydrolysates of Colocasia esculenta (Taro) leaves. Number in the figure shows the PO4-P concentration (mg/g). Vertical bars show the error in estimation as standard deviation. For acetic acid treatment, nitrate-N (Figure 7) was found to be 0.01–0.02 mg/g of bio-mass for both 30 min and 1 h reaction times. TKN was estimated to be 2.05 mg/g of bio-mass for the 5% treatment for 30 min. There was an insignificant change in the nitrate Figure 8. Pho phate-P (mg/g) in different hydrolysates of Co oc sia esculenta (Tar ) leaves. Numberin the figure shows the PO4-P concentration (mg/g). Vertical bars show the error in estimation asstandard deviation.F r acetic acid treatment, nitrate-N (Figure 7) was found to be 0.01–0.02 mg/g fbiomass for both 30 min and 1 h reaction times. TKN was estimated to be 2.05 mg/gof biomass for the 5% treatment for 30 min. There was an insignificant change in theSustainability 2022, 14, 16383 15 of 18nitrate recovery with changes in the concentration and time. For ferric chloride treatment,the nitrate-N (Figure 7) values decreased with an increase in the chemical concentrationirrespective of the time parameter. For the 30 of treatment, nitrate-N was 0.02, 0.01, and0 mg/g of biomass for 1%, 2%, and 5% concentrations, respectively. For the 1 h treatment,the corresponding values were 0.01, 0, and 0 mg/g of biomass. TKN was measured for 1 hof treated hydrolysate and it was found to be 1.2, 2.5, and 5.08 mg/g of biomass for 1%,2%, and 5% concentrations. Soluble TKN increased due to the production of intermediatecompounds such as protein, amino acids, and other nitrogenous components [56].Nitrate-N (Figure 7) for nitric acid treatment was higher than the other chemical treatmentdue to the presence of nitrate in the hydrolyzing reagent itself. For the reference treatment (1%and 2% H2SO4), nitrate-N was between 0.02 and 0.04 mg/g of biomass for 30 min and 1 h,respectively. TKN was approximately 2.54 mg/g of biomass for 1% treatment for 30 min, andfor the 2% treatment, TKN was 4.72 mg/g of biomass for the 1 h reaction.The solubilization of orthophosphate-P (Figure 8) for acetic-acid-treated hydrolysateswas higher than the other chemical treatments and ranged from 0.88–1.27 mg/g of biomass.There was a significant change in the solubilization of phosphate with an increase in theconcentration. For ferric-chloride-treated hydrolysates, orthophosphate-P solubilizationwas found to be much lower, and the content decreased with an increase in the concentration(Figure 8). Ferric chloride leads to the formation of Iron phosphate and its oxyhydroxidecomplexes, and these complexes have their minimum solubility at a pH between 4 and 6.This could be the reason behind the low phosphorus recovery in the hydrolysate preparedfrom both biomasses. To remove phosphorus through chemical precipitation, one needs tomaintain alkalinity, pH, and hardness at the proper level. These are the main controllingfactors behind phosphorus precipitation [40].The orthophosphate-P (Figure 8) content was between 0.29 and 0.9 mg/g of biomassfor nitric-acid-treated hydrolysates. There was an insignificant change in the phosphaterecovery with a change in concentration and time for nitric-acid-treated hydrolysates.The orthophosphate-P (Figure 8) release for 1% H2SO4 treatment was 0.24 and 0.25 for30 min and 1 h reactions, respectively. The corresponding values for 2% H2SO4 treatmentwere 0.39 and 0.69 mg/g of biomass, respectively. According to Kim et al. [57], usingchemicals for hydrolysis procedures can also form trace amounts of soluble hydroxide andphosphate complexes. However, the concentrations of these complexes did not interferewith precipitation. To understand the attractiveness of each biomass, the carbohydrate, N,and P release from the best hydrolyzing reagent were compared. It was observed that foralgae, the carbohydrate release was less than that of Taro leaves (a ratio of algae: Taro of0.44). However, the release of N and P was much higher for algal biomass (4 to 11 timeshigher in algae). Hence, it can be deduced that Taro leaves may be a good source of sugarscompared to algae, whereas algae would be the best source to recover nutrients.4. ConclusionsAlgae and Colocasia esculenta (Taro) leaves can be utilized as low-cost waste biomassto recover sugars and nutrients. Among the chemicals used for hydrolyzing the biomass,different chemicals acted differently for the respective biomass. Acetic acid was foundto be the best for algal biomass hydrolysis with a sugar yield of 44.2 mg/g biomass(solubilized monomers—0.82 mg/g of biomass), and for Colocasia esculenta (Taro) leaves,ferric chloride treatment was the best with a sugar yield of 95 mg/g biomass (solubilizedmonomers—43.6 mg/g of biomass). PO4-P release from algal biomass was significantlyaffected by the concentration of the hydrolyzing agents. Statistical models revealed that inmost cases, an increase in concentration would also increase the P release. However, forcarbohydrate release, in most cases, no such significant effect was observed. The statisticalmodel indicated that carbohydrate release from Taro leaves was affected by the time andconcentration for most of the chemical hydrolyzing agents used in this study. Based onthe results of this study, in the future, composting followed by chemical treatment will beexplored to improve the sugar and nutrient yield.Sustainability 2022, 14, 16383 16 of 18Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su142416383/s1, Figure S1: Carbohydrate (mg/g) in the differenthydrolysates of pure cellulose powder. Number in the figure shows the concentration (mg/g) of individualcomponents (lagend). Number on the top of each column shows the total carbohydrate content; Table S1: CODand TOC data obtained from various hydrolysis procedures for algae, Table S2: Characteristics ofhydrolysate prepared from algae using various hydrolysing agents.Author Contributions: Conceptualization, S.D. and R.C.; methodology, S.D., R.C., S.G. and P.K.;formal analysis, S.D. and R.C., investigation, S.D. and R.C.; resources, P.K. and S.G.; data curation,R.C. and S.D.; writing—original draft preparation, S.D.; writing—review and editing, S.D., R.C. andA.S.; visualization, S.D. and R.C.; supervision, R.C.; project administration, P.K. and R.C.; fundingacquisition, R.C. and P.K. All authors have read and agreed to the published version of the manuscript.Funding: The research work is a collaborative study between the University of British Columbia,Canada (funding agency: IC-IMPACT) and Indian Institute of Technology, Roorkee, India, (Fundingagency: Dept. of Biotechnology, Govt. of India).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: Not applicable.Conflicts of Interest: The authors declare no conflict of interest.References1. Show, K.Y.; Yan, Y.; Ling, M.; Ye, G.; Li, T.; Lee, D.J. Hydrogen production from algal biomass-advances, challenges and prospects.Bioresour. Technol. 2018, 257, 290–300. [CrossRef] [PubMed]2. Dahiya, S.; Chowdhury, R.; Tao, W.; Kumar, P. Biomass and Lipid Productivity by Two Algal Strains of Chlorella sorokiniana grownin Hydrolysate of Water Hyacinth. Energies 2021, 14, 1411. [CrossRef]3. 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chemical hydrolysis
algae
Colocasia esculenta (Taro) leaves
carbohydrate
nutrient solubilization
acidic treatment
Recovery of Sugar and Nutrients from Algae and Colocasia esculenta (Taro) Leaves Using Chemical Hydrolysis
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