3.1. Corn Steep Water Composition
The composition of CSW results in the extraction of water-soluble components during the steeping step of corn, as shown in Table 1
. The CSW is mainly composed of carbon (around 37%) and nitrogenous (about 7%) components (elemental analysis data not shown), being the total carbon from organic sources. Regarding organic acids, lactic acid is the most predominant one (6.7 g/L), followed by the phytic acid (3.1 g/L) and acetic acid (2.8 g/L). Monosaccharides, like glucose (0.3 g/L) and arabinose (0.2 g/L), as well disaccharides such as sucrose (0.04 g/L) are part of the CSW composition. Physiologically relevant ions such as chloride, phosphate, sulphate, ammonium, calcium, boron, iron, potassium, magnesium, sodium, manganese, zinc, and silicon, were detected in CSW at substantial quantities. Among them, potassium (0.8 g/L) and phosphate (0.5 g/L) are the most significant; while aluminum, chromium, copper, cobalt, and nickel are the less represented. Hull and Montgomery [32
] suggested that the high amount of inorganic phosphate in CSW could be due to in part to being a product of dephosphorylation of myo-inositol phosphates.
The above composition agrees with the composition analyzed by Hull et al. [33
] at various times during the steeping of corn and from four different industrial processes. However, the novelty of this composition is the concentration of biosurfactant. This bioactive compound is present in a concentration of 2 g/L, based on the CMC data of biosurfactant extracted from con steep water by liquid-liquid extraction with chloroform (see Figure S1
). This finding, related with the use of CSW as a direct source of biosurfactants, and its uses have been previously patented and published by Vecino et al. [1
]. Nowadays, depending on the industrial application of the biosurfactant extract from CSW, an organic solvent is proposed for the extraction process. However, the possibility of using a liquid-solid extraction process by calcium alginate-based biopolymers to recover biosurfactants from CSW has not been considered until now.
3.3. Calcium Alginate-Based Biopolymers Performance in Liquid-Solid Process to Recover Biosurfactants from Corn Steep Water
In this study, two types of calcium alginate-based biopolymers were formulated without and with the presence of grape marc for the recovery of biosurfactants in CSW. Table 2
shows the removal percentage of the inorganic solutes and biomolecules, that comprise CSW, after adsorption process for a contact time for 24 h (e.g., equilibrium was attained), using alginate biopolymer and alginate biopolymer with grape marc.
The biopolymer that only contains alginate presented a high capacity for the elimination of organic acids and sugars from the CSW, with a maximum removal for acetic acid (100%) and glucose (98.5%), respectively. For almost all ions, metals as well as biosurfactant extract, the percentage of removal was about 50%, except for boron (40.4%) and silicon (39.2%). On the other hand, the alginate-based biopolymer tunned with grape marc, also showed good elimination rates for the same families, organic acids, and sugars, but for other types. For instance, the highest percentage of elimination was for lactic acid (99%) and for all the sugars tested this was greater than 99%. Moreover, this biopolymer had obtained good removal of metals such as iron (98.5%) and zinc (96.8%) as well as for cations, like manganese (90.4%), and anions, such as phosphate (75%). However, the amount of biosurfactant extract eliminated was of 55%.
Overall, the combination of calcium alginate-based biopolymer with grape marc provided better results in comparison with only calcium alginate biopolymer; except for boron and silicon, where only the alginate biopolymer offered greater affinity for these compounds. The initial hypothesis is supported by the fact that grape marc possesses lower adsorption capacity for these elements than calcium alginate, which reduces the adsorption capacity for boron and silicon. When the bio-adsorbent is formulated with sodium alginate, the active surface sites for adsorption are based only in the calcium alginate-based polymer, whereas when the bioadsorbent is formulated with sodium alginate (2%) plus grape marc (2%), the active surface sites, for adsorption, based on calcium alginate, were lower, since the latter biopolymer has a lower percentage of alginate, with respect to the total composition, than the former. Additionally, the adsorption of biosurfactant extract was not greatly influenced by the type of biopolymer since there was only around 8% difference between them. Indeed, it is important to remark that this is a multi-adsorption mechanism, occurring simultaneously with physical and/or chemical adsorption and ion-exchange processes. Moreover, the corn stream is composed of several inorganic solutes and biomolecules that interact between them, adding more complexity to the adsorption process. However, in a previous study [30
], a calcium alginate-based biopolymer tunned with grape marc was evaluated for the removal of color in wastewater. It was observed that the adsorption process followed a pseudo-second order kinetic model (heterogeneous process). In addition, in another physicochemical study with a bio-based adsorbent made from grape marc [35
], the physical control of the sorption stage was well- described by the Freundlich isotherm.
Biodegraded grape marc entrapped in calcium alginate beads has been previously tested as an eco-adsorbent. For instance, Perez-Ameneiro et al. evaluated the use of a biopolymer based on grape marc entrapped in calcium alginate beads for the removal of pigments from an agro-industrial effluent [30
] or dye compounds from winery wastewater [35
] as well as to remove micronutrients from winery effluents in order to avoid eutrophication [36
]. Additionally, an alginate-based polymer with grape marc has also been tested as an eco-adsorbent for removal of copper (II) from aqueous streams [37
]; for adsorption of binary mixtures of dyes [38
]; and for the removal of cyanide and transition metals from industrial electroplating process waters [39
]. Nevertheless, it should be highlighted that this type of biopolymer has never been tested to recover biosurfactants present in corn steep water.
In the case study of micronutrients [36
], the calcium alginate-based biopolymer with grape marc removed most of the TN, NH4+
existing in the vinasses (wastewater from winery industry) and about 60% of Mg, P, K, and total carbon. Similar results were obtained in the current study, with 66% of Mg, 75% of P, 55% of K and 49% of TOC. It is important to note that in the corn steep water, the concentration of Mg and P was almost ten times more, being double for K and around six times more for the total carbon than vinasses.
On the other hand, the adsorption capacities for the inorganic solutes and biomolecules tested using calcium alginate-based biopolymers, after 24 h of equilibration time, are displayed in Table 3
The maximum adsorption capacity was achieved for lactic acid with both biopolymers, which was 167.9 and 332.4 mg/g for the calcium alginate-based biopolymer without and with grape marc, respectively. This maximum capacity is followed by the capacity for acetic acid (141.1 mg/g) and phytic acid (114.2 mg/g) in the case of the biopolymer alone with alginate; and this order of adsorption capacity value is reversed in the biopolymer containing grape marc: phytic acid with 129.7 mg/g and acetic acid with 118.2 mg/g. Additionally, both biopolymers presented high capacity for TOC between 102.9 mg/g (alginate) and 136.9 mg/g (alginate plus grape marc); and the capacity of TN was 35.3 and 47.6 mg/g for the alginate biopolymer and the alginate biopolymer tunned with grape marc, respectively. Concerning the biosurfactant adsorption capacity, both biopolymers showed values of 46.8 and 54.8 mg/g for the alginate biopolymer and the alginate biopolymer tunned with grape marc, respectively. For the other inorganic solutes and biomolecules, that compose the CSW, the calcium alginate-based biopolymers provided adsorption capacities values below 24 mg/g.
Depending on the solute and its concentration, the capacity of calcium alginate-based biopolymer, tunned with grape marc, changes substantially. For example, in the study of pigment removal from vinasses [30
], the capacity of the biopolymer varied between 0.28 and 0.76 mg/g as a function of the initial dye concentration (8.8 to 24.7 mg/L). Also, in the case of mixtures of dyes [38
], the adsorption capacity was 2.47 and 2.22 mg/g for methylene blue and methyl red, respectively, using calcium alginate-based biopolymer with grape marc; whereas, the biopolymer based only in calcium alginate achieved capacity values lower than 2 mg/g. These capacities are very low compared to those obtained in this work. However, the biopolymer capacities for metal ions were higher. Pérez-Cid et al. [39
] concluded that calcium alginate hydrogel beads can be considered a bioadsorbent with a high capacity to remove free cyanide (1177 mg/g) and transition metals (Ni, Cu and Zn as follows 107.3, 39.5 and 1.52 mg/g, respectively) in electroplating streams. In this case, the introduction of composted grape marc in the calcium alginate bead formulation did not produce significant improvements in the adsorption capacity. Comparatively, the Zn adsorption capacity, achieved in the current work, was higher 23.9 mg/g, but the initial concentration of zinc in the corn stream was around ten times less and also lower quantity of adsorbent was used (the CSW/biopolymer ratio used was 1:1 (v/v
) vs. the wastewater/bioadsorbent ratio used was 1.5:1 (v/v
)) than in the previous study. Otherwise, the maximum adsorption capacity was reached by using the calcium alginate-based biopolymer with grape marc for copper (II) removal from sulfate solutions [37
]. In this study, the adsorption capacity value was around 1800 mg/g after 5 min of contact time, while it increased to 2785 mg/g at the highest concentration assessed (0.15 mol/L) and at the maximum extraction times (20 min).
3.4. Characterization of Corn Steep Water before and after Adsorption Processes
The corn steep waters were characterized based on CMC, FTIR and ESI-MS analysis. Raw corn steep liquor possesses a CMC of 10–15 g/L depending on the provider company, providing minimum surface tension values to water between 36.6 and 46.7 mN/m [1
]. However, these results are not comparable with those obtained in this work since the corn steep water has been centrifuged (for solids removal) and lyophilized. Thus, in the current work the CMC of the corn steep water, as shown in Figure 2
, was 0.46 g/L, reaching a minimum surface tension of 54.5 ± 0.2, observing that solids increased the CMC of raw corn steep liquor, although they provide a higher reduction of surface tension in water.
The concentration at which the micellization starts is known as CMC. At this concentration not only the micelles are made, but also the lower surface tension value of the solution is achieved [40
]. Thus, after the adsorption processes, with the calcium alginate-based biopolymers, the CMC values increased almost two-fold, being 0.99 and 1.15 g/L for the biopolymer based on calcium alginate and calcium alginate tunned with grape marc, respectively; whereas the minimum surface tension remained constant (54.2–55.3 mN/m). In this case, although the minimum surface tension value had been kept constant, an increase in CMC implies that a higher amount of biosurfactant extract is needed to reach this value of surface tension. Therefore, the corn steep water reduced its surfactant capacity, since the biosurfactant extract is adsorbed by these calcium alginate-based biopolymers. Thus, CMC results are in concordance with the percentage removal of the biosurfactant as a function of the type of biopolymer (see Table 2
). Therefore, greater capacities for biosurfactants obtained with the biopolymers, involved higher CMC values for the treated corn steep water. Although the presence of biosurfactants in those streams treated with calcium alginate polymer tunned with grape marc was lower.
On the other hand, Figure 3
shows the FTIR spectra of the initial corn steep water (grey line) as well as the corn streams after adsorption processes with the calcium alginate-based biopolymers (orange and blue lines). Also, it was used to determine the similarities of the corn stream before and after the adsorption processes.
The FTIR spectrum of the biopolymer only with alginate is in concordance with the results of characterization carried out by Aburabie et al. [28
], where the absorption region of stretching vibrations of O–H bonds (3344 cm−1
) in calcium alginate membranes was narrower and more elongated than sodium alginate solution; while the C–O stretching vibration (1080 and 1021 cm−1
) decreased in calcium alginate membranes.
In general terms, the bands of corn stream that are affected, after the adsorption process, correspond to the functional groups of proteins, lipids, and polysaccharides. The characterization was as follows: (i) the wavenumbers between 3400 and 3000 cm−1
corresponded with the peptide groups resulting from O–H and N–H stretching; (ii) bands around 3000 and 2000 cm−1
as well as 1400 cm−1
related to the presence of C–H stretching corresponding to aliphatic chains (CH2
groups) of fatty acids; (iii) the bands C=O bond at 1640 cm−1
(amide I bond) and N–H bonds at 1540 cm−1
(amide II bond) indicated the presence of protein-related weakness; and iv) the band around 1000 cm−1
denoted the presence of polysaccharides, conforming the C–O stretch vibration [41
The bands of the three main functional groups (proteins, lipids, and polysaccharides) are decreased after the adsorption process with both calcium alginate-based biopolymers. This is confirmed by the degree of similarity of the initial corn stream before and after adsorption processes, which was 69.6% and 72.1% for the biopolymer alone with alginate or alginate tunned with grape marc, respectively between 400–4000 cm−1
. It would be expected that the corn stream treated with the alginate-based biopolymer tunned with grape marc would be more different from the initial corn stream, since the removal rates for most of the inorganic solutes and biomolecules were higher than those obtained after treating the corn steep water with the biopolymer only based on calcium alginate (see Table 2
). Additionally, the biosurfactant extract obtained from corn steep liquor is a lipopeptide [1
]; therefore, observing the protein band, it would be expected that the biopolymer with grape marc would produce a greater reduction in the band of corn steep water, due to the higher removal percentage of biosurfactant achieved in comparison with the biopolymer only formulated with calcium alginate (around 55% vs. 47%). This finding could be due to the fact that the calcium alginate-based biopolymer tunned with grape marc provided nitrogen to corn steep water, since grape marc is a biodegradable material with the following composition: 3.9% N, 42.7% C and 5.5% H [30
Moreover, the ESI-MS analysis allowed us to corroborate the adsorption of certain inorganic solutes and biomolecules such as the biosurfactant by the biopolymers under evaluation. Figure 4
shows the natural molecular masses of the bioactive molecules present in the raw corn stream as well as those present in the corn steep water after adsorption treatment with the calcium alginate-based biopolymers. The following signals were detected in raw corn steep water: at 175, 219, 263, 330, 423, 493, 570, 659, 806, 879, 966 as well as signals between 1200–1300 m/z. Some of them, for instance 879, were m/z also detected in the biosurfactant extract obtained from corn steep liquor, after the liquid-liquid extraction process, in previous studies [43
]. In fact, the biomarker observed at 879 m/z (and at 617 m/z) was derived from the fragmentation of the signal observed at 933 m/z, with signals compatible with the presence of lipopeptide biosurfactants [43
]. Additionally, Li et al. [44
] suggested that the presence of triglycerides, diglycerides and monoglycerides could be signals below 800 m/z. Ma et al. [45
] detected, in the ESI-MS/MS spectrum, at 441 m/z, several surfactin precursors. Nevertheless, in the biosurfactant extract from CSW, obtained after liquid-liquid extraction with organic solvents, this signal could correspond to the presence of phenolic compounds [46
Otherwise, signals within the range of 800 and 1200 m/z are mass biomarkers of biosurfactant precursors produced by Bacillus
strains widely described in lipopeptide analyses [44
]. Thus, the masses higher than 800 m/z relatively disappeared from the ESI spectrum, in treated corn steep water, corroborating that part of the biosurfactant extract was trapped by the calcium alginate-based biopolymers.