*Article* **Evaluation of the Use of Recycled Vegetable Oil as a Collector Reagent in the Flotation of Copper Sulfide Minerals Using Seawater**

**Felipe Arcos and Lina Uribe \***

School of Mining Engineering, University of Talca, Talca 3460000, Chile; farcos14@alumnos.utalca.cl **\*** Correspondence: luribe@utalca.cl; Tel.: +56-220-1798

**Abstract:** Considering sustainable mining, the use of seawater in mineral processing to replace conventional water is an attractive alternative, especially in cases where this resource is limited. However, the use of this aqueous medium generates a series of challenges; specifically, in the seawater flotation process, it is necessary to adapt traditional reagents to the aqueous medium or to propose new reagents that achieve better performance and are environmentally friendly. In this research, the technical feasibility of using recycled vegetable oil (RVO) as a collector of copper sulfide minerals in the flotation process using seawater was studied. The study considered the analysis of the metallurgical indexes when different concentrations of collector and foaming reagent were used, considering as collectors the RVO, potassium amyl xanthate (PAX) and mixtures of these, in addition to the methyl isobutyl carbinol (MIBC) as foaming agent. In addition, it was evidenced that the best metallurgical indexes were achieved using 40 g/t of RVO and 15 g/t of MIBC, which corresponded to an enrichment ratio of 6.29, a concentration ratio of 7.01, a copper recovery of 90.06% and a selectivity index with respect to pyrite of 4.03 and with respect to silica of 12.89. Finally, in relation to the study of the RVO and PAX collector mixtures, it was found that a mixture of 60 g/t of RVO and 40 g/t of PAX in the absence of foaming agent presented the best results in terms of copper recovery (98.66%) and the selectivity index with respect to pyrite (2.88) and silica (14.65), improving PAX selectivity and recovery compared to the use of RVO as the only collector. According to these results, it is possible to conclude that the addition of RVO improved the selectivity in the rougher flotation for copper sulfides in seawater. This could be an interesting opportunity for the industry to minimize the costs of the flotation process and generate a lower environmental impact.

**Keywords:** flotation; seawater; collectors; vegetable oil; recycled vegetable oil

#### **1. Introduction**

Froth flotation is one of the most widely used physicochemical methods of concentrating minerals in the mining industry today because of the high capacity and efficiency it offers in the process of obtaining metals [1]. Separation of minerals by froth flotation techniques depends primarily on the differences in the wettability of particles. The most important reagents used are the collectors, which selectively adsorb onto the surface of the mineral particles, making them hydrophobic and facilitating their adhesion to gas bubbles injected into the process that rise through the slurry to form a concentrate rich in the mineral of interest [2].

Among the different collectors currently available in the copper mining industry, the xanthates are widely used in the flotation of minerals such as common sulfides (Cu, Mo, Pb, Zn, Co and Ni) and native metals (Cu, Ag, Au, etc.). The xanthates are ionic collectors and contain a hydrophilic polar group (affinity with water) attached to a chain of hydrocarbons that turns out to be the hydrophobic part. Due to their strong ionic powers, xanthates have negative effects to the biota and health hazards to human and animals [3–5], and they become a major problem at the time of handling and disposal of the waste generated by

**Citation:** Arcos, F.; Uribe, L. Evaluation of the Use of Recycled Vegetable Oil as a Collector Reagent in the Flotation of Copper Sulfide Minerals Using Seawater. *Recycling* **2021**, *6*, 5. https://doi.org/ 10.3390/recycling6010005

Received: 14 December 2020 Accepted: 4 January 2021 Published: 11 January 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the flotation [6]. It was estimated that a total of 52,000 tons of xanthates were consumed annually by the mining industry worldwide [7]. Most of the mining industry across the globe use xanthate. The efforts to reduce the harmful effects of these are focused on the search to develop a "green" collector, or environmentally friendly, that reduces or inhibits the generation of toxic substances.

On the other hand, because of the water scarcity in some mining areas around the world, some projects consider the use of seawater to process coper ores by flotation in countries such as Chile, Australia and Indonesia [8]. In Chile, Las Luces plant has been using sweater for their operations since 1994 [9], and more recently Esperanza mine from Antofagasta Minerals [10]. However, even though the use of seawater in mineral process can be seen as interesting alternative to solve the scarcity problems, its use in mineral processing leads to significant challenges such as corrosion in different equipment such as pipes, mills and flotation cells; and the reduction of the performance in the concentration process [11]. Within this framework, as possible solutions to these problems, it has been proposed to use some coatings of materials with high chemical and mechanical resistance [12], to use alloys of materials that better resist the corrosion phenomenon [13] and to study new operating conditions in the different processes associate, in order to achieve metallurgical recoveries and grades of copper without affecting the efficiency of by-products (Mo and Au) [14].

Specifically, when seawater is used to carry out this concentration process, a series of problems is generated due to the high concentration of ions that it possesses (Na<sup>+</sup> , K<sup>+</sup> , Mg2+, Ca2+, SO<sup>4</sup> <sup>2</sup><sup>−</sup> and Cl−). Mainly, there is the pH adjustment as seawater acts as a buffer solution, which generates a significant increase in lime consumption to achieve the pH values required in the process to depress pyrite [15]. Additionally, seawater generates a depressant effect on minerals such as molybdenite at pH values above 9.5. This is mainly due to the presence of calcium and magnesium cations in solution, which generates colloidal precipitates at pH levels above this value, preventing the recovery of this mineral [16]. It is because of these reasons that, new reagents are needed, in order to recover copper minerals (Chalcopyrite) in seawater, without affecting the recovery of secondary minerals, such as molybdenite. In addition, these new reagents must be able to depress pyrite to the pH of seawater.

In this sense, the use of vegetable oils could be an interesting alternative to replace and/or reduce the use of xanthates in copper sulfide ore flotation. There exists evidence that shows that the collecting properties of these oils can be competitive compared to the commercial collectors used in the sulfides flotation and native minerals [17–21]. Owusu et al. studied the use of commercial edible oils (canola, derived from rapeseed, and palm oil) as potential flotation reagents to add to the existing array of xanthates normally deployed for the selective flotation of copper sulfide minerals [18]. In addition, Bauer et al. [20] and Greene et al. [21] patented the use of various oils as collectors for the recovery of chalcopyrite and molybdenite from an ore containing 0.579% Cu and 0.010% Mo. Furthermore, Benn et al. [22] proposed the use of rapeseed oil as a selective collector for galena (PbS) recovery against pyrite. A laboratory flotation test using a complex sulfide ore containing Pb, Zn, Cu and Fe sulfides using 240 g/t of rapeseed oil as collector gave a higher recovery of lead, copper and zinc minerals than when typical, conventional collectors (xanthate, dithiophosphate, mercaptan, etc.) were used. Pyrite recovery in this study was similar to that obtained using the conventional collectors.

Taking into account these considerations, this research proposes to study a recycled vegetable oil as a collector of copper sulfide minerals in the flotation process in seawater in order to contribute to reduce the toxic collector used in minerals processing and to find a different type of application of this waste This study considered the analysis of the metallurgical indexes when different concentrations of collector and foaming reagent were used, considering as collectors the RVO, potassium amyl xanthate (PAX) and mixtures of these, in addition to the methyl isobutyl carbinol (MIBC) as foaming agent.

#### **2. Material and Methods**

#### *2.1. Mineral Sample*

The mineral sample used in this investigation was supplied by Minera Paicaví S.A. from the Chépica mine, located in the town of Pencahue, Chile. For the purposes of this work, each sample of 600 g of ore were crushed, ground in a ball mill and sieved to a P80 size of 212 µm, which corresponds to an appropriate size for rougher flotation.

Tables 1 and 2 present the results of the XRF and XRD analysis performed on the mineral sample. In the first one, it can be observed that, according to XRF analysis, the copper content was 2.20% and iron was 12.97%, while, regarding its mineralogy, in the second table, it is observed that the content of these elements are mainly associated to chalcopyrite and pyrite and that gangue minerals are mainly quartz and phyllosilicates such as muscovite and chlorite.


**Table 1.** Results of the XRF analysis of the mineral sample.

**Table 2.** Results of the XRD analysis of the mineral sample.


#### *2.2. Chemical Reagents and Aqueous Media*

The collector reagents used in this research were potassium amyl xanthate (PAX) supplied by SOLVAY Chile and purified according the methodology described by Montalti et al. [23]. Sunflower recycled RVO was collected by Universidad de Talca and was previously filtered and was introduced in the flotation cell as supplied. The foaming agent used was methyl isobutyl carbonyl (MIBC Aerofroth 70 supplied by SOLVAY, Antofagasta, Chile). The only modifier considered in this project was lime (CaO) to adjust the pH to 8. MIBC was made down to 1 mL/L, by weighing 1 g into a clean 100 mL volumetric flask and making up to the mark with distilled water.

The supply of seawater used in this research was from the coast of the Maule region in Chile, specifically from the town of Iloca, highlighting that the water was extracted at a point far from the shore so that its chemical composition was not affected or physically contaminated. The collected seawater had a pH of 7.6 and salinity of 35 g/L. During the flotation stage, all experiments were done at room temperature of 20 ◦C.

#### *2.3. Experimental Procedure*

#### 2.3.1. Reduction of Mineral Sample Size

The first comminution process was performed in the jaw crusher in order to obtain a P80 size of 2000 µm. Then, a wet milling process was performed, for which 67% of solids by weight (using 600 g of ore sample and 300 g of water) ore slurry was prepared. A first addition of reagents was made during this stage, specifically of lime (0.7 g) to regulate the pH to the desired value (pH 8). In addition, to determine the optimum grinding time, kinetic tests were carried out beforehand, allowing the grinding time to be set at 50 min.

#### 2.3.2. Flotation Test

The flotation process was carried out in a Edemet cell of 1.5 L by adding a pulp containing 600 g of mineral and 1200 g of water, forming 33% of solids by weight, maintaining a pH 8, adding wash water after each palletization and dosing the collecting and foaming reagents according to the test to be carried out for the investigation. The reagents were added at the same time during conditioning stage, which lasted 5 min and was done at an agitation speed of 1100 rpm. At this stage, the pH value was also adjusted to the desired value by the addition of lime. Once this was achieved, the flotation stage took place, which lasted 16 min and was carried out at a stirring speed of 1000 rpm, injecting an airflow of 5 L/min maintaining a manual palletization every 10 s. All tests were done in duplicate with an average standard error of 5%.

Finally, once each flotation test was completed, both the concentrate and the process tail were dried. First, an Edemet pressure filter was used to remove the excess water and then the samples obtained were dried inside the drying oven for approximately 24 h at a temperature of 100 ◦C. Subsequently, the corresponding analyses were carried out using XRF to determine the content of Cu, Fe, S and Si in each sample and finally to calculate the metallurgical indexes for each test.

Rougher flotation tests were performed by evaluating the dosages of RVO or PAX collector to 20, 40, 60, 80 and 100 g/t. After that, mixtures of RVO/PAX at 80/20, 60/40, 40/60 and 20/80 g/t, respectively, were evaluated. Finally, to evaluate the effect of MIBC frother in the presence of RVO, the best dosages of collectors (PAX or RVO) or mixtures of them were kept constant, according to the metallurgical results, to evaluate the dosages of MIBC at concentration levels between 0 and 20 g/t.

2.3.3. Calculations and Methods of Analysis Used

• Metallurgical Indexes

The most important metallurgical indexes studied were the grade and the recovery of Cu and Fe in the concentrate, since these data provide an indication of the efficiency of the flotation. Similarly, enrichment ratio, selectivity indexes, concentration ratio and concentration yield were studied. Table 3 shows the formulas used to calculate these indexes.


**Table 3.** Formulas to calculate metallurgical performance [24].

• Applied Assumptions

To analyze the amount of chalcopyrite and pyrite present in the concentrates, the following mathematical relations were made based on the stoichiometric composition of chalcopyrite and pyrite, after having obtained the percentage of copper and iron in the concentrate by means of XRF.

This application is based on the assumption that all the copper present is associated with the chalcopyrite ore using a certain amount of iron. On the other hand, the remaining iron content is related to pyrite [18].

Equation (6) is used to estimate the percentage of chalcopyrite present in the concentrate.

$$\% \mathbb{C} \, py = \% \mathbb{C}u \left(\frac{183.3}{63.5}\right) \tag{6}$$

Equation (7) refers to the percentage of iron present as pyrite as estimated above.

$$\%Fe^\* = \%Fe - \%\text{Cu} \left(\frac{55.8}{63.5}\right) \tag{7}$$

Finally, Equation (8) represents the percentage of pyrite present in the concentrate.

$$\%Py = \%Fe^\* \left(\frac{119.8}{55.8}\right) \tag{8}$$

#### **3. Results and Discussion**

#### *3.1. Results of Collective Flotation Tests as a Function of PAX and RVO Concentration*

Figure 1a,b shows the results obtained from the recovery and grade of copper and iron, as a function of PAX (Figure 1a) and RVO (Figure 1b) concentration. Figure 1a clearly shows that, in the absence of PAX, the lowest recovery and highest copper grade is obtained, which was to be expected due to the fact that under these conditions only the most hydrophobic particles of the ore, such as copper sulfides, should float. Subsequently, the addition of 20 g/t of PAX collector produces an increase in recovery because this reagent gives hydrophobic characteristics to the ore particles, achieving at 40 g/t the highest recovery and copper grade. Finally, at higher concentrations of this reagent, it can be seen that recovery and copper grade decrease considerably, suggesting an overdose of collecting reagent Accordingly, it should be noted that a 40 g/t concentration of PAX collector allows for the best copper recoveries and grades in seawater, under the operating conditions set forth above. On the other hand, in relation to the recovery and iron grades obtained at different PAX concentrations, a similar trend can be observed in the curves obtained with copper, suggesting that the iron present in the concentrate is mainly associated to chalcopyrite and pyrite minerals.

Figure 1b shows the results obtained for recovery and grade of copper and iron, as a function of the different studied RVO collector concentrations. In the figure, it can be seen that, in the case of copper, the recovery curve shows a significant increase when RVO is added to the process, regardless of its concentration, going from having a recovery of 58.8% in the absence of collector reagent to achieving an average recovery of close to 90% at the different RVO concentrations studied. Similarly, when analyzing the copper grade, it is observed that, at concentrations between 20 and 80 g/t of RVO collector, the highest copper grades are achieved, which is close to 9%, while, in the absence of collector and at a maximum concentration of 100 g/t, this grade decreases to values of 6.84% and 5.77%, respectively. On the other hand, with respect to the recovery and grade of iron, it is possible to note that, at 80 g/t, the highest recovery and grade was achieved with 65.7% and 29.42%, respectively.

**Figure 1.** Recovery and grade of copper and iron obtained based on the PAX (**a**) and RVO (**b**) concentration, using 15 g/t of MIBC and seawater.

It is important to point out that, within the interval between 20 and 80 g/t of RVO, we have both the highest grades and the highest copper recoveries obtained with this collector, considerably improving the results obtained in its absence, which indicates that this reagent has a positive effect on the process and a great potential to be considered as a collector reagent in the flotation of copper sulfides with seawater.

Figure 2a,b shows the recovery and grade of both minerals (Cpy and Py) obtained as a function of the PAX and RVO concentration, respectively. In the figures, it can be seen that, for both minerals and collectors studied, there is a similar trend to the grades and recoveries obtained from each element, with the highest recoveries and grades of chalcopyrite at a concentration of 40 g/t. However, when comparing the results obtained in the presence of 40 g/t of PAX and RVO separately (Figure 2a,b), it is noteworthy that the chalcopyrite grades in the presence of RVO (28.2%) are significantly higher than those obtained with PAX (19.4%). On the other hand, it is also shown that pyrite grades remain between 40% and 45% and the increase in the concentration of the RVO collector does not significantly affect the grade of this mineral. On the basis of the above, it can be stated that the same amount of pyrite is concentrated in the presence of RVO as with PAX, but there is a considerable increase in the percentage of chalcopyrite. The above indicates that RVO adsorbs preferentially on copper particles and that in seawater this reagent delivers favorable results in terms of both recovery and copper grades.

**Figure 2.** Recovery and grade of chalcopyrite and pyrite obtained based on the PAX (**a**) and RVO (**b**) concentration, using 15 g/t of MIBC and seawater.

Tables 4 and 5 show the metallurgical indexes calculated for the PAX and RVO collectors in flotation tests. Within these indexes, we find the recovery; grade; enrichment ratio; selectivity index with respect to copper–silica and chalcopyrite–pyrite, because these

species are the main gangue that are required to be removed during the flotation process; and the concentration ratio and yield.

**PAX Dosage (g/t) R (%) Cu Grade (%) Re IS (Cu-Si) IS (Cpy-Py) Rc V** 0 58.78 6.84 4.69 6.27 1.95 8.02 0.12 20 95.41 4.41 2.99 14.92 2.06 3.15 0.32 40 96.85 6.70 1.93 7.62 1.98 1.99 0.50 60 82.32 2.85 1.93 3.42 1.36 2.36 0.42 80 56.40 2.45 1.47 1.65 1.05 2.63 0.38 100 54.05 2.29 1.34 1.47 1.10 2.48 0.40

**Table 4.** Metallurgical indexes obtained based on the PAX concentration, using 15 g/t of MIBC and seawater.

**Table 5.** Metallurgical indexes obtained based on the RVO concentration, using 15 g/t of MIBC and seawater.


From the results presented in Table 4, it can be seen that the best metallurgical indexes with respect to copper were achieved at 20 and 40 g/t of PAX collector, being 20 g/t the case in which the highest enrichment ratio, concentration ratio and selectivity of copper with respect to the main gangue, silica and pyrite and 40 g/t the case in which the highest recovery and grade of copper is achieved. With regard to the capacity indexes obtained at these conditions, it is noted that the highest concentration ratio occurs at 20 g/t and, consequently, the highest concentration yield at 40 g/t. On the other hand, in relation to the RVO collector from the results presented in Table 5, it can be seen that the best metallurgical indexes with respect to copper were achieved at 40 and 80 g/t of RVO collector, being 40 g/t the case in which the highest grade, enrichment ratio, concentration ratio and selectivity of copper with respect to the main gangue, silica and pyrite and 80 g/t the case in which the highest copper recovery is achieved.

Comparing the results obtained in the presence of PAX and RVO, it is possible to establish that, at 40 g/t of RVO collector, the flotation process achieves better metallurgical indexes than those obtained with PAX, in which it can be clearly indicated that the copper grade, the enrichment ratio, the selectivity indexes and the concentration ratio are better when using RVO than PAX, being the copper recovery the only index favored in the presence of PAX.

#### *3.2. Results of Collective Flotation Tests Using Mixtures of RVO/PAX Collectors in Different Proportions*

Tests were performed by mixing both collector reagents with the aim of studying possible configurations of the collectors that would have favorable effects on metallurgical indexes.

Figure 3 shows, in the same way as the previous cases, the recovery curves and copper and iron grades obtained as a function of the different RVO/PAX mixtures studied. In the figure, it can be seen that, in the case of copper, the recovery obtained for the 80/20, 60/40, 40/60 and 20/80 reagent mixtures is approximately constant, reaching a recovery close to 98%. Meanwhile, in cases where separate reagents were used, it can be seen that the

recovery of copper is lower with 100 g/t of PAX than when using the same concentration of RVO, with 88.80% and 54.05%, respectively. By analyzing the copper grade, it can be seen that the highest grades are obtained in the 80/20 and 60/40 RVO/PAX blends, which corresponded to 5.51% and 7.40%.

**Figure 3.** Recovery and grade of copper and iron obtained based on the RVO/PAX concentration, using 15 g/t of MIBC and seawater.

On the other hand, with respect to the recovery and iron grades obtained as a function of the RVO/PAX mixtures, it can be seen that the curves show a behavior similar to that of copper, reaching a recovery for the 80/20, 60/40, 40/60 and 20/80 cases of approximately 90%, and, with respect to the iron grade, it can be seen that the highest grades of this element are obtained in the 80/20 and 60/40 RVO/PAX mixtures, which were close to 30%.

When comparing the results of the recoveries and grades obtained in the mixtures with those obtained only with RVO (Figure 1b), it can be seen that, in the mixture, the presence of PAX favors the recovery of copper obtained with RVO but affects the copper grade obtained in the concentrate.

With respect to the mineralogy associated with the process, Figure 4 shows the recovery and grade of chalcopyrite and pyrite obtained for each mixture according to the assumptions indicated above. In the figure, it can be seen that the highest recovery and chalcopyrite grade was obtained with an RVO/PAX mix of 60/40, obtaining 98.6% and 21.4%, respectively.

Comparing the results obtained with the mixtures against those obtained only with RVO (Figures 2 and 4), it can be observed that the presence of PAX generates a slight increase in the recovery of chalcopyrite but a decrease in the grade of this mineral and, in the same way, significantly increases the presence of pyrite in the concentrates, being the mixture RVO/PAX 60/40 the one that presents the best results.

Table 6 presents the metallurgical indexes calculated for the flotation test set performed. It shows that, in general, the best metallurgical indexes with respect to copper were achieved with the RVO/PAX 60/40 mixture, especially in terms of process selectivity.

**Figure 4.** Recovery and grade of chalcopyrite and pyrite obtained based on the RVO/PAX concentration, using 15 g/t of MIBC and seawater.

**Table 6.** Metallurgical indexes obtained based on the RVO/PAX concentration, using 15 g/t of MIBC and seawater.


When comparing the results under this condition (Table 6) with the best results obtained with the separate action of each collector (Tables 4 and 5), it can be established that the use of reagent mixtures improves the copper recovery compared to that obtained with both collectors separately, while the grade and the enrichment ratio were improved when compared with PAX alone. On the other hand, compared to the selectivity indexes with respect to the main gangue, it can be seen that the selectivity of copper, with respect to silica, increased when using mixtures, while, with respect to pyrite, it was only increased when comparing it with PAX. Finally, in. relation to the concentration ratio, the use of mixtures increased this index with respect to the value obtained with PAX, but, when compared with those obtained with RVO alone, these values are significantly higher, generating the concentrates with the lowest amount of mass in the presence of RVO.

In general, the reagent mixtures generated a positive effect, achieving 98.24% and 7.40% maximum copper recovery and grade, respectively, when using an RVO/PAX 60/40 mixture. Although it is true that the grade is higher than that obtained with PAX and lower compared with the cases of RVO, the results obtained with this mixture with respect to the other metallurgical indexes are attractive considering that the studied process corresponds to a stage of collective flotation.

#### *3.3. Seawater Flotation Results as a Function of MIBC Concentration*

3.3.1. Results of Collective Flotation Tests as a Function of MIBC Concentration in Presence of 40 g/t PAX and RVO, Separately

Figure 5 presents the results of copper and iron recovery and grade obtained as a function of MIBC concentration. In the figure, it is possible to observe that the recovery curves for both elements showed a similar trend and a constant behavior when varying the MIBC concentration, while, in the case of copper and iron grades, they increase as the concentration of the foaming agent increases. A minimum recovery of copper in the absence of foaming agent is obtained, corresponding to 96.6% and a maximum of 97.8% at 20 g/t MIBC. On the other hand, in relation to the recovery of iron, a behavior similar to that of copper is obtained, with a minimum recovery of 89.7% in the absence of foaming agent and a maximum recovery of 90.8% using 10 g/t of MIBC. With respect to the grades, in the case of copper, the lowest value was 5.43% and occurs at 5 g/t MIBC, from which it begins to increase progressively and slightly depending on the concentration of foaming agent, reaching the maximum value of 7.23% at 20 g/t. On the other hand, when analyzing the iron grade obtained in the concentrate, it is emphasized that there is a similar behavior to the previous one, where the lowest grade obtained for iron was 23% at 5 g/t MIBC, while the highest was 28.7% at 20 g/t foaming agent. In this way, and according to the previous evidence, it can be established that for this test with 40 g/t of PAX collector, the best results in terms of grade and copper recovery are obtained with 20 g/t of MIBC foaming agent.

**Figure 5.** Recovery and grade of copper and iron obtained based on the MIBC concentration, using 40 g/t PAX and seawater.

Figure 6 shows the recovery and grade curves of copper and iron obtained at the different concentrations of foaming agent for a 40 g/t RVO concentration. In the figure, it is possible to emphasize that the recovery of copper has a stable behavior that oscillates between 87% and 90% in the interval that goes from 0 to 15 g/t, presenting the minor recovery at 20 g/t, which was of 84.7%. On the other hand, in relation to the recovery of iron, it can be seen that the curve obtained showed a behavior similar to that of copper, i.e., recovery remains stable at around 56%, while at 15 g/t, unlike the case of copper which slightly increased its recovery, for iron it decreases markedly to around 44% approximately. In relation to the grades obtained as a function of the MIBC concentration, in the case of copper, it can be observed that in the absence of foaming agent a grade of 9.74% is achieved, which then, by adding low concentrations of foaming agents, corresponding to 5 and 10 g/t, decreases to values of 8.33% and 7.92%, respectively. However, at higher concentrations, corresponding to 15 and 20 g/t, a considerable increase is achieved, reaching values of 9.74% and 9.51%, respectively. With

respect to the iron grade, a similar behavior can be seen to that obtained in the case of copper, which at 0 g/t has a value of 28.37%; at concentrations of 5 and 10 g/t decreases to 27.79% and 27.26%, respectively; and at higher concentrations the grade is reversed, obtaining the highest value at 15 g/t of MIBC, corresponding to 28.79%.

**Figure 6.** Recovery and grade of copper and iron obtained based on the MIBC concentration, using 40 g/t RVO and seawater.

On the other hand, Figure 7a,b shows the percentages of chalcopyrite and pyrite obtained as a function of the concentration of foaming agent. It shows a similar behavior to that obtained with copper and iron grades, where, as the concentration of foaming agent increases, the grades of both minerals increase, the highest grades being obtained at a dose of 20 g/t MIBC, 20.9% for chalcopyrite. With regard to the recovery of these minerals, this index shows little variation depending on the concentration of the foaming agent, reaching a maximum value of 98.3% at 20 g/t of MIBC, thus improving the results obtained before 15 g/t.

In addition, Figure 7b shows the recovery and grade of chalcopyrite and pyrite obtained in the presence of RVO. It can be seen that chalcopyrite is directly related to copper grade, so at 0 and 15 g/t it has its highest grade with a value of 28.2%. On the other hand, it is observed that the pyrite grade in the concentrate remains practically constant between 42% and 43%. With regard to the recoveries of both minerals, it is observed that they remain practically constant throughout the range of foaming agent concentrations studied, obtaining the highest recovery of chalcopyrite at 15 g/t MIBC with a value of 98.2%, while the lowest recovery of pyrite is reached at 20 g/t.

Table 7 presents the metallurgical indexes obtained as a function of the concentration of foaming agent. It shows that, at 20 g/t MIBC, the best metallurgical indexes are indeed obtained. It should be pointed out that the Cu-Si selectivity index increases considerably in the presence of this dose of MIBC, reaching 11.99, as well as for the Cpy-Py case, where a value of 2.43 is obtained. Finally, with respect to the capacity indexes, the concentration ratio is higher in comparison to the other MIBC concentrations, indicating that the concentrate obtained in the process has a lower mass quantity, making the process more efficient in both collective and selective terms. Consequently, the lowest concentration yield is obtained of all the conditions studied, confirming the fact of having a lower mass quantity in the concentrate with respect to the process feed. On the other hand, in relation to the RVO collector from the results presented in Table 8, it is observed that the best indexes are obtained at a concentration of 15 g/t, highlighting both the enrichment ratio of 6.29, the selectivity index Cu-Si of 12.89 and Cpy-Py of 4.03, as well as the concentration ratio of

7.01, indicating a high selective capacity in the process in this condition of operation and dosage of reagents in the presence of seawater.

**Figure 7.** Recovery and grade of chalcopyrite and pyrite obtained based on the MIBC concentration, using 40 g/t PAX (**a**) and RVO (**b**), separately, and seawater.


**Table 7.** Metallurgical indexes obtained based on the MIBC concentration, using 40 g/t PAX and seawater.

**Table 8.** Metallurgical indexes obtained based on the MIBC concentration, using 40 g/t RVO and seawater.


In the same context, the selectivity index of chalcopyrite on pyrite obtained at 15 g/t MIBC stands out due to its high value, which was 4. According to the literature, this represents a good metallurgical result for the flotation process, which, integrated with the rest of the indexes studied, makes this case a good operating condition in terms of feasibility for its use in industrial processes. Finally, the concentration yield is highlighted, which indicates that, of the total mass fed to the flotation, only 14% is transferred to the concentrate stream, suggesting high selectivity for the process.

3.3.2. Results of Collective Flotation Tests as a Function of MIBC Concentration in Presence of RVO/PAX 60/40 Mixture

Figure 8 presents the results of copper and iron recovery and grade obtained as a function of MIBC concentration using a 60/40 g/t RVO/PAX mixture as collector. In general, it is possible to observe that all the curves obtained have a practically stable behavior as a function of the MIBC concentration. With regard to copper recovery, it can be seen that its behavior is homogeneous throughout the whole range of frother agent concentration, i.e., it remains constant with a value close to 98%. On the other hand, the recovery of iron also behaves in a stable way, maintaining a recovery that ranges between 90.78% and 92.4%.

With respect to the grades obtained, it can be observed that, with respect to the copper grade, it remains stable with a value close to 7%, reaching its maximum value in the absence of MIBC corresponding to 7.58% of copper in the concentrate and a minimum value at 5 g/t corresponding to 7.05%. On the other hand, analysis of the iron grade may indicate that it exhibits constant behavior as a function of the MIBC concentration, the maximum and minimum iron grade being obtained at 15 and 5 g/t, respectively, with values of 29.49% and 28.80% for each of the aforementioned doses.

**Figure 8.** Recovery and grade of copper and iron obtained based on the MIBC concentration, using RVO/PAX 60/40 and seawater.

Figure 9 shows the grade and recovery of chalcopyrite and pyrite obtained at the different concentrations of foaming agent studied. It is possible to observe in the figure that the highest chalcopyrite content, as well as the highest copper grade, is achieved in the absence of foaming agent and corresponds to 21.9% together with 48.5% pyrite. Meanwhile, the lowest grade of the latter ore was 47.7% and is reached at a concentration of 10 g/t MIBC, which coincides with one of the lowest values for iron grade. With regard to the recovery of these minerals, in general, it is maintained without great alterations depending on the concentration of the foaming agent; however, the maximum recoveries of chalcopyrite are reached at 0 and 5 g/t with values of approximately 98.9%, while the maximum recovery of pyrite occurs at 5 g/t with a value of 92.5%.

**Figure 9.** Recovery and grade of chalcopyrite and pyrite obtained based on the MIBC concentration, using RVO/PAX 60/40 and seawater.

Table 9 presents the metallurgical indexes obtained according to the different concentrations of foaming agent. It shows that, with respect to copper recovery and grade, there is a very slight variation between each test depending on the concentration of the foaming agent and the highest result is obtained in the absence of MIBC. Compared to the enrichment ratio, a similar behavior is observed, with the highest enrichment at 15 g/t MIBC. On the other hand, compared to the selectivity indexes, the Cu-Si selectivity index presented its highest values at 5 g/t of frother, while for the Cpy-Py case the highest value was obtained in the absence of MIBC. Finally, in relation to the capacity indexes, it is observed that the highest concentration ratio was achieved in the absence of foaming agent along with the best performance.

**Table 9.** Metallurgical indexes obtained based on the MIBC concentration, using RVO/PAX 60/40 and seawater.


From the results obtained for the different concentrations of MIBC for the best separate collectors (Tables 7 and 8) and the mixture of these (Table 9), it can be seen that, except for recovery, the best metallurgical indexes obtained are obtained when using 40 g/t of RVO and 15 g/t of MIBC. On the other hand, the use of RVO/PAX mixtures allows the flotation process to be carried out in the absence of foaming agent, achieving even better metallurgical indexes than those obtained with conventional PAX collectors. This phenomenon could be explained by the presence of RVO in the process, which could play an additional role as frother, given its physical and chemical properties. In this aspect, it is necessary to emphasize that the use of mixtures achieves a great performance in collective terms, achieving the highest recovery of all the conditions studied and the highest selectivity with respect to the Cu-Si case, as well as a mass reduction of approximately four times of the feed to the concentrate stream.

#### **4. Conclusions**

This research proposed to study an RVO as a collector of copper sulfide minerals in the flotation process in seawater in order to contribute to reducing the toxic collector used in minerals processing and to find a different type of application of this waste. Based on the metallurgical analysis, it is possible to note that the RVO collector was more selective for chalcopyrite than PAX but gives a lower recovery. In addition, by analyzing the mixture of both collectors, in the absence of frother, the best results in collective terms and selectivity were obtained. According to these preliminary results, it is possible to conclude that the addition of RVO improved the selectivity of the conventional collector PAX, which is highly collective, in the rougher flotation of copper sulfide minerals in seawater. This could be an interesting opportunity for the industry in order to minimize the costs of the flotation process and generate a lower environmental impact and significant health advantages since it could help to reduce the use of toxic xanthates. However, future research is needed in order to find the best method of addition of the RVO to the process, to study new mixtures with other collectors and to analyze the frother characteristics. In addition, it could be important to understand the effect of the presence of RVO in subproducts as molybdenite.

**Author Contributions:** Conceptualization, methodology, data curation, visualization, validation, formal analysis, investigation, writing-original draft preparation writing-review and editing, F.A. and L.U. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors wish to thank the support from the ANID/FONDAP/15130015 project.

**Conflicts of Interest:** The authors declare not conflict of interest.

#### **References**


## *Review* **Prospects of Red King Crab Hepatopancreas Processing: Fundamental and Applied Biochemistry**

**Tatyana Ponomareva, Maria Timchenko , Michael Filippov, Sergey Lapaev and Evgeny Sogorin \***

Federal Research Center "Pushchino Scientific Center for Biological Research of the RAS", 142290 Pushchino, Russia; tatyanap91875@gmail.com (T.P.); matimchenko@gmail.com (M.T.); filippmv@gmail.com (M.F.); a3t1v4i1n5@gmail.com (S.L.)

**\*** Correspondence: evgenysogorin@gmail.com; Tel.: +7-915-132-5419

**Abstract:** Since the early 1980s, a large number of studies on enzymes from the red king crab hepatopancreas were conducted. They have been relevant both from a fundamental point of view in terms of studying the enzymes of marine organisms and in terms of rational natural resource management aimed to obtain new valuable products from the processing of crab fishing waste. Most of these works were performed by Russian scientists due to the area and amount of waste of red king crab processing in Russia (or the Soviet Union). However, the close phylogenetic kinship and the similar ecological niches of commercial crab species and the production scale of the catch provide the bases for the successful transfer of experience in the processing of the red king crab hepatopancreas to other commercial crab species caught worldwide. This review describes the value of recycled commercial crab species, discusses processing problems, and suggests possible solutions for these issues. The main emphasis is made on hepatopancreatic enzymes as the most salubrious products of red king crab waste processing.


**Citation:** Ponomareva, T.; Timchenko, M.; Filippov, M.; Lapaev, S.; Sogorin, E. Prospects of Red King Crab Hepatopancreas Processing: Fundamental and Applied Biochemistry. *Recycling* **2021**, *6*, 3. https://dx.doi.org/10.3390/ recycling6010003

Received: 19 October 2020 Accepted: 29 December 2020 Published: 2 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** commercial crab species; red king crab; waste processing; hepatopancreas; proteases; hyaluronidase

#### **1. Introduction**

The global growth in consumer demand for food products based on commercial species of marine organisms (fish, crab, squid, etc.) has stimulated fishers to increase production. However, the inability to increase the catch volume endlessly and related waste recycling problems have put forward the consideration of advanced processing of secondary raw materials to obtain new commercially valuable products. Crabs are a favorite catch of fishers worldwide due to the high price of their meat. The main northern commercial species of crab include the following: Red king crab (*Paralithodes camtschaticus*), Blue king crab (*Paralithodes platypus*), Spiny brown king crab (*Paralithodes brevipes*), Golden king crab (*Lithodes aequispinu*), Opilio snow crab (*Chionoecetes opilio*), Tanner snow crab (*Chionoecetes bairdi*), Triangle tanner crab (*Chionoecetes angulatus*), Red snow crab (*Chionoecetes japonicus*), Chinese mitten crab (*Eriocheir sinensis*), Hair crab (*Erimacrus isenbeckii*) [1]. The first four species belong to the infraorder of half-tailed or false crabs (Anomura) of the suborder Pleocyemata, order Decapoda. The rest of them belongs to the infraorder of true crabs (Brachyura) (Figure 1) [2].

**Figure 1.** Phylogeny of some members of the class Malacostraca. Species with known primary structures of hyaluronidases are underlined (see Section 3.4.3).

All crabs have a massive cephalothorax covered with carapace from above, a flat abdomen, and are bent under the cephalothorax. Brachyura representatives move with the help of four pairs of pectoral legs, and the fifth pair of limbs are claws. A specific feature of the Anomura is the asymmetric structure of the body (the right claw is larger than the left) and the presence of only three pairs of walking legs (one of the five pairs is hidden under the carapace and is used for the regular cleaning of gills) [1,3–5].

Red king crab, or Kamchatka crab, is famous around the world. In recent decades, the catch of this crab by Russian fishers has reached 15,000–20,000 tonnes per year. The original habitat of red king crab ranges from Karaginsky Island (Russia) off the east coast of Kamchatka and Shelikhov Bay (Russia) in the Sea of Okhotsk to Hokkaido Island (Japan) and the Korea Strait (between Korea and Japan). The crab is also found on the west coast of North America from Cape Barrow to the Queen Charlotte Archipelago in the south [1,6].

Red king crab was successfully introduced to the Barents Sea in the 1960s and 1970s. Optimal temperature conditions, the absence of natural predators, and sufficient amount of food led to the spread of acclimatized red king crab from the coast of the Kola Peninsula to Norway and north to Svalbard (Figure 2). Individuals of this king crab population are larger, grow faster, and mature earlier than individuals of the far-eastern population [7]. The rapid growth of the northern king crab population is an environmental problem [7,8]. The growth rate of the resource potential of this population made it possible to open commercial fishing in Norway in 2002 and in Russia in 2004.

**Figure 2.** Red king crab native and invasive distribution.

In Russia, the crab catch is often processed immediately on board. One of the methods for processing the crab on a ship is as follows: the just-caught crab is placed on a hook; the limbs are additionally cleaned, boiled, and frozen; and the broken carapace with entrails is dumped immediately into the sea as a waste product. On average, this waste can account for 50% of the catch mass [9,10]. The mass fraction of carapace from these wastes is approximately 60%; the rest comprises the entrails (including the digestive organ, the hepatopancreas) [6]. In terms of protein content, these wastes are identical to crab meat, and even superior in terms of the content of minerals, lipids, and carbohydrates. Crab flour used as animal feed can be obtained from crab processing waste. In addition, the crab shell is an excellent source of raw materials for the production of chitin and chitosan, which can be used to meet the demands of the food industry and medicine [10]. Chitin, chitosan, and their derivatives represent promising matrices for the development of novel biomaterials with antioxidant, bactericidal, hypotensive, antiallergic, antiinflammatory and antitumor activities [11]. Recently, crab shell has also been used for the production of *α*-glucosidase inhibitors and anticancer agent prodigiosin via microbial fermentation [12,13]. Due to their peculiar properties such as nontoxicity, biocompatibility, and biodegradability, chitin and chitosan are used as potential excipients and as biological active agents in cosmetology [14].

The main focus of the review is on the research and use of the crab hepatopancreas enzyme complex due to the strong industry interest in this enzyme source. Russia is the leader in the catch of this fishing object. This is reflected in the number of studies on the processing of this waste by Russian scientists. Most of these works were published only in Russian, which significantly limited the availability of information on that research results and development for the world community. The goal of this review is to solve this problem.

#### **2. Materials and Methods**

The review has been systematically approached based on electronic databases including Scopus, Web of Science, Google Scholar. Most of the resources are the articles of highly specialized journals without translation and abstracts of regional symposia and conferences specialized in the water resources of Russia (Soviet Union), which are not available in electronic form.

Figure "Red king crab native and invasive distribution" was generously provided by GRID-Arendal (nonprofit environmental communications centre based in Norway). This graphic item may be reproduced in any form for educational or nonprofit purposes without special permission from GRID-Arendal, provided acknowledgement of the source is made [15,16].

The chapter "Technologies for processing hepatopancreas" presents patented technologies for processing waste from red king crab and other closely related commercial crabs. This information corresponds to the study of the technical level of patent research on this topic.

Experimental data from research articles were used to compile tables. Among other things, the molecular masses of the proteins under study and the methods used in these works are indicated. The table shows the scatter of these data from different works in order to demonstrate the measurement by different researchers. The correct molecular weight should be estimated by the results of mass spectrometry and interpretation of the results of calculations for the amino acid sequence of proteins.

#### **3. Results**

#### *3.1. Crab Hepatopancreas*

The hepatopancreas is an organ of the digestive system that functions as both the liver and pancreas [17]. In red king crab, the hepatopancreas makes up about 90% of the intestines of the carapace and 5–10% of the total weight of the animal [18]. The Decapoda hepatopancreas secretes a wide variety of highly active enzymes.

Under the action of the digestive enzymes of the hepatopancreas, food is broken down into easily digestible substances. The hepatopancreas of crabs of the family Lithodidae (infraorder Anomura, for example, red king crab) consists of brown mass of fragile liver tubules filling the main part of the body cavity. The integrity of these tubules is easily destroyed, even by a slight mechanical action, and the enzymes enter the body cavity, where they start the process of autolysis. The hepatopancreas of true crabs (Brachyura, for example, opilio crab) is shapeless orange-brown mass [1].

#### *3.2. Use of Red King Crab Hepatopancreatic Enzymes*

The hepatopancreas of the digestive system of commercial crabs is a valuable source of a complex of enzymes with various activities: collagenase, protease, hyaluronidase, lipase, nuclease, etc. The complex of proteolytic enzymes of the red king crab hepatopancreas is of interest in various industries. For example, the prospect of using hepatopancreas enzyme preparation in the hydrolysis of soy protein was recently highlighted [19]. A red king crab hepatopancreas enzyme preparation was successfully used to separate roe from the connective tissue of ovaries of commercial fish [20], to hydrolyze minced fish meat to obtain a dietary food ingredient [21], to hydrolyze crustacean processing waste products to obtain components for microbiological nutrient media [22], and to isolate chondroitin sulfate from marine wastes, namely from tissues of marine organisms [23]. Based on collagenolytic proteinases, wound healing and wound cleansing preparations [24–26], including wound dressings [27,28], have been designed.

#### *3.3. Hepatopancreas Recycling Technologies*

Many technologies are used to process the hepatopancreas of commercial crab species to obtain enzymatic preparations. Most technologies were developed for the hepatopancreas of red king crab, but some were transferred to the processing of the hepatopancreas from other commercial species (for example, snow crab and blue crab) [29,30]. Most often, the initial raw material is the hepatopancreas (Figure 3), which is homogenized in salt buffers or by osmotic shock under hypotonic conditions (excess distilled water). The integrity of the hepatopancreas tissue is easily destroyed during the freezing/thawing process; therefore, no considerable effort is required for mechanical homogenization. However, in some cases, a colloid mill can be used [31]. Sometimes, triton X-100 or sodium

dodecyl sulfate (SDS) is added to the homogenization buffer [29,32]. The homogenate is incubated at room temperature for several hours. Under such conditions, cell autolysis occurs, which increases the level of protein extraction; however, this can lead to the inactivation of target enzymes. A significant problem in further processing is caused by lipids. Ballast substances and lipids are removed from the homogenate by centrifugation or flocculation followed by chitosan filtration [30–34] or immediately by filtration through the hollow fiber [35,36]. The filtrate is concentrated by ultrafiltration through hollow fibers with a pore size of 15–30 kDa and then dried by freeze-drying or spray-drying. The choice of hollow fibers with a specific pore size (considering the variation in real pore sizes) should be based on the known molecular weights of the target enzymes. To obtain more purified preparations before drying, proteins are precipitated with ammonium sulfate and/or tert-butanol, and then ion exchange, hydrophobic, or affinity chromatography is performed [37–40]. In the simplest case, after homogenization, proteins are precipitated with an excess of cooled acetone ("acetone powder") [41]. This method is not suitable for upscaling, since the use of a large volume of acetone is unsafe for both humans and the environment. Most of the developed technologies have been successfully tested on tens and hundreds of kilograms of hepatopancreas as raw materials [29–31,33–36]. The yield of dry final substance in such protocols varies from 0.6 to 1.3% based on the feedstock [33–36], and the collagenolytic activity is 500–3000 units Mandl/mg (the substrate is collagen type I, at 37 ◦C) [35,36], even reaching 11,000 Mandl/mg (the substrate is collagen type III, at 42 ◦C) [29], whereas the amount of protein in dry matter is 80–98% [29,33,35,36].

The basic conditions for enzymes obtained with the mentioned technologies include the maintenance of neutral pH of the enzyme solution; at pH values below 5.5 and above 9.5, the activity of the proteolytic complex and its individual components decreases dramatically. At pH values below 3 and above 10.5, parts of the enzymes are irreversibly inactivated [33,42].

**Figure 3.** Concept of hepatopancreas processing.

#### *3.4. Enzymes of the Red King Crab Hepatopancreas*

#### 3.4.1. Proteolytic Enzymes

Currently, 10 proteolytic enzymes of hepatopancreas—collagenolytic serine proteinase PC (PC—*Paralithodes camtschaticus*), collagenolytic serine proteinase PC 2, trypsin-like proteinase A, chymotrypsin-like proteinase C, aminopeptidase PC, carboxypeptidase PC, trypsin PC, elastase, cathepsin L, and metalloproteinase—were described in the literature in detail (Table 1). Most of these are small proteins up to 30 kDa (based on the denaturing gel electrophoresis data) with an isoelectric point (pI) of 2.5–4.4. The optimal operating conditions for these enzymes include neutral pH and temperature range from 25 to 55 ◦C. The collagenolytic serine proteinases are of particular interest due to their ability to degrade native collagen of types I–III. Most likely, these two enzymes are the basis of the active substance in existing wound healing and wound cleaning preparations. Collagenolytic activity has also been assigned to other hepatopancreas proteinases. However, the temperature of the reaction mixture in these studies was often 42 ◦C, at which native collagen partially denatures [43]; therefore, the measured activity may refer not to true collagenase but to gelatinase activity. Despite the large number of scientific works devoted to hepatopancreas enzymes, the complete nucleotide sequence of mRNA is known only for collagenolytic serine proteinase PC, trypsin PC, cathepsin L, and metalloproteinase (Table 1).

**Table 1.** Hepatopancreas proteolytic enzymes. <sup>1</sup> expected molecular weight based on the mRNA nucleotide sequence, <sup>2</sup> mass spectrometry data. PC—*Paralithodes camtschaticus*.



**Table 1.** *Cont.*

3.4.2. Nucleases and Other Enzymes of Hepatopancreas

The red king crab hepatopancreas is a source of enzymes such as nucleases (Table 2). Ca2+- and Mg2+-dependent DNase, which is characterized by high thermal stability, has been studied in detail: the enzyme remains absolutely active after 3 h of incubation at 60 ◦C, whereas incubation for 30 min at 100 ◦C resulted in only 7% loss of activity [60]. Based on the circular dichroism (CD) spectrum, the protein appears to be a compact globule and consists mainly of *β*-layers (75%) [61]. The pI value is 4 and the optimal reaction temperature range is 50–60 ◦C [62].

This DNase has a pronounced specificity for the secondary structure of DNA and predominantly cleaves double-stranded substrates (DNA and RNA–DNA duplexes, while the RNA remains intact). The minimum duplex size for cleavage by DNase is 9 bp; the enzyme does not hydrolyze RNA [62]. The DNase cleaves phosphodiester bonds with the formation of 5′ -phosphate and 3′ -OH terminal groups. Ca2<sup>+</sup> and Mg2<sup>+</sup> together have a positive synergistic effect on the rate of the hydrolysis reaction. The unique properties of DNase enable it to be used effectively for the rapid analysis of single nucleotide polymorphisms

and the normalization of nucleic acid libraries [63–66]. In the hepatopancreas of red king crab, other nucleases (RNases) and phosphoesterases were found (Table 2).

**Table 2.** Nucleases and other enzymes of hepatopancreas. <sup>1</sup> denaturing gel chromatography data, <sup>2</sup> denaturing electrophoresis, <sup>3</sup> expected molecular weight based on the mRNA nucleotide sequence.


The enzyme preparation from the hepatopancreas contains several other enzymatic activities, which allows this enzyme complex to be used for the depolymerization of *β*glycosidic bonds in chitosan [69–71]. Lipase activity has also been observed [33]; however, other activities of hepatopancreas enzymes have not been sufficiently studied in comparison with proteolytic and nuclease activities.

#### 3.4.3. Hyaluronidase Activity of Hepatopancreas Homogenate

Currently, the structure of hyaluronidases of the Malacostraca class has not been studied in sufficient detail. In the open database of UniProt protein sequences, there are only four representatives of this class for which amino acid sequences of hyaluronidases are available: two representative of Decapods, including Whiteleg shrimp (*Litopenaeus vannamei*, UniProt A0A423SH46) and orange mud crab (*Scylla olivacea*, two UniProt proteins A0A0P4VVV1 and A0A0N7ZAX3), and two representative of Isopoda, which are pill-bug *Armadillidium vulgare* (only one of two proteins is characterized as UniProt A0A444ST78) and *Armadillidium nasatum* (UniProt A0A5N5TJL6) (Figure 1). Earlier, hyaluronidase

activity was found in the complex of enzymes of the red king crab hepatopancreas homogenate [72].

In our previous work, the kinetics of the hydrolysis of hyaluronic acid in cosmetic fillers with the red king crab hepatopancreas homogenate was studied for the first time [73]. Using the methods of turbidimetric analysis, atomic force microscopy, and nuclear magnetic resonance spectroscopy, the kinetics of hydrolysis and the structural transformation of hyaluronic acid under the action of homogenate enzymes were investigated. We found that the obtained homogenate has activity comparable to commercially available hyaluronidase preparations. In this work, we demonstrated the possibility of using an enzymatic preparation based on hepatopancreas homogenate for the treatment of complications after the injection of fillers based on hyaluronic acid. Further studies of the effectiveness and safety of hyaluronidase from hepatopancreas on model animals will enable us to develop new drugs for the treatment of complications of filler injections in the near future.

#### *3.5. Other Valuable Non-Protein Components of the Red King Crab Hepatopancreas*

The crab hepatopancreas is a source of enzymes and other valuable products. For example, a preparation with the properties of an inhibitor of serine proteinases was obtained and characterized from the hepatopancreas, and its effect on the process of human blood plasma coagulation was studied [74]. The tested inhibitor showed an anticoagulant effect that was more pronounced when combined with heparin. Procedures hwere developed to obtain an inhibitor both from the raw material [75] and in the recombinant form [76].

The hepatopancreas contains a large amount of fat, which varies from 10 to 26% [18,77,78]. In the study of the fractional composition of crab fat, it was found to contain triglycerides at a rate of up to 55%, as well as polyunsaturated fatty acids, including *ω*-3 (14–24% of the total of all fatty acids). Hepatopancreas fat does not contain toxic substances and can be used as a food supplement or as an ingredient for cosmetic products.

#### **4. Discussion**

#### *4.1. Prospects of Processing Waste from Other Commercial Crab Species*

The commercial species of crabs include representatives of the false (Anomura) and true crabs (Brachyura) of Pleocyemata. Brachyura includes opilio crab (*Chionoecetes opilio*), which is also a commercially important catch for marine fisheries. Crabs of this suborder have similar enzymatic activity in their digestive system. For example, the proteolytic activities of enzyme preparations from the hepatopancreas of red king crab and opilio crab are practically the same [21]. The zymogram showed that the hepatopancreas of opilio crab contains at least 10 proteolytic enzymes.

The activity of the proteolytic complex was comparable to the commercially available collagenase of the gas bacillus *Clostridium histolyticum* [79,80]. In these works, proteolytic enzymes of the hepatopancreas of opilio crab, but not red king crab, were isolated and biochemically characterized, and the N-terminal amino acid sequences of proteolytic enzymes were obtained. The authors noted their mistake in their next work [42]. Unfortunately, these incorrect data were published in the UniProt database. For example, the amino acid sequence UniProtKB-P20734 (COGC\_PARCM) actually belongs not to *Paralithodes camtschaticus*, but to *Chionoecetes opilio*. The sequence of UniProtKB-P34153 (COG1\_CHIOP) and UniProtKB-P34156 (COG4\_CHIOP) is not derived from the hepatopancreas proteins of *Chionoecetes opilio*, but from *Paralithodes camtschaticus*. The rest of the N-terminal sequences of proteolytic enzymes of these two crabs in [79,80] completely coincide in the UniProt database, which once again confirms the biochemical similarity of the digestive systems of the representative of Pleocyemata.

The close phylogenetic kinship and similar ecological niches of the commercial crab species, as well as the industrial scale of the catch, provide grounds for the successful transfer of the experience of the processing of the king crab hepatopancreas to other crab species to obtain new valuable products. For example, the enzymatic complex of hepatopancreas of opilio crab was successfully used in the production of protein hydrolysates from cod

waste processing, as well as to improve the consistency and juiciness of cod fillets at the stage of the salting of the semi-finished product [21,30,81].

Russia is a leader in terms of the catch level of red king crab, and this catch is increasing every year (Figure 4a). Most of this catch comes from fishers in the Russian Far East, however, the total catch of Russia and Norway in the Barents and Norwegian Seas is expected in the near future to equal and even exceed the catch level in the Russian Far East [82]. In the United States (Alaska), red king crab is also caught, but to a lesser extent. Fishers in Alaska have caught 200–400 tonnes per year from 2013 to 2020 [83]. However, at the same time, the United States catches about 5000–10,000 tonnes of crabs that are collectively known as king crabs [82]. Other commercial species of crabs are also promising species for advanced processing. For example, Food and Agriculture Organization (FAO) data indicate that the global catch of opilio crab is more than 100,000 tonnes [84] (Figure 4b). We confidently state that high catch levels of large commercially significant crabs, and therefore vast amount of waste, provide an opportunity to develop waste processing technologies and include them in the industrial process. The most promising direction of processing should be considered the processing of the hepatopancreas as a source of new high-margin products.

**Figure 4.** Crab catch: (**a**) red king crab in the Barents Sea (Russia and Norway) and the Russian Far East (Russia) and (**b**) the global catch of opilio crab.

#### *4.2. Development Strategy for Waste Processing*

Crab is processed using different approaches. If the caught crab is not boiled, complex waste processing can be used to maximize the yield of new valuable products. After limb separation, the rest of the body can be completely processed: the carapace can be used for chitosan production, the hepatopancreas for the production of multicomponent enzymatic complexes and specific purified enzymes, the fat from the hepatopancreas for dietary nutrition or biofuel production, and the gills and other internal organs as a feed supplement for birds, fish, and other animals. This approach adopts the principle of non-waste recycling.

The methods of processing the crab hepatopancreas to obtain enzyme preparations can be divided into two strategies: obtaining complexes of various enzymes or further purification of specific enzymes (or class of enzymes) from this complex. The enzyme complex is appropriate for the treatment of multicomponent substrates where the simultaneous degradation of proteins, nucleic acids, and other polymers into monomers is required, for example, in the production of easily digestible food products from animal and plant tissues. The activity of the enzyme complex in the preparations will differ for

the hepatopancreas from sample to sample. In this regard, the technology for using such enzymatic preparations should allow for fluctuations in the activities in different batches of the preparation. The second strategy, based on the isolation of one enzyme from the complex, has advantages, since the final product can be used to produce a high-margin product (for example, a pharmaceutical product or a reagent for scientific research). For this purpose, it is important to have a simple and scalable method for the purification of a specific enzyme. Notably, all the currently existing technologies for processing the crab hepatopancreas do not meet the specified requirements. The main vector of development in this field could be an approach including several stages of tangential flow filtration and affinity chromatography using a cheap carrier.

All these works discussed in this review consider the processing of a specific type of waste (carapace/hepatopancreas/fat). However, the waste is most often a mixture, which is difficult to sort. To implement the principle of non-waste recycling, it is necessary to develop new approaches to recycling this mixture, which will convert the waste into new valuable products. The relevance of deep processing is provided by the sustainable continual increase in catches of fisheries. On the one hand, a growth in catch leads to an increase in waste, which might cause several environmental problems. On the other hand, insufficient control of the catch will lead to a decrease in the crab population, which will entail a sharp decline in the economic performance of the industry in the coming years. Thus, all types of processing could reduce the environmental burden, as well as help satisfy the financial appetites of the industry by selling new valuable products.

The main problem with the complex processing of wastes from commercial marine species is the complexity of their collection and storage under sailing conditions on board a trawler and delivery to coast or further processing. The solution of this problem could be, for example, the creation of a maximally automated integrated waste processing unit immediately on a catching vessel, which could eliminate the problem of the time spent by fishers on the manual removal of an organ, as well as its storage and transportation. Another method of solving this problem is to establish an adequate cost of the hepatopancreas for fishers, allowing a stable supply of raw materials onshore on an industrial scale. The task could be simplified if the non-waste processing of live crabs is organized onshore. Further studies of the hepatopancreas of commercial crab species in fundamental scientific terms considering its potential applications will increase the value of crab processing waste, possibly leading to a time when this will equal or even exceed the cost of crab meat.

#### **5. Outlook**

Despite a long period of scientific research, deep processing of crab has not been launched yet. The main reason for this is laboratory protocols being not adapted to catch conditions. Also these technologies should provide deep recycling based on the principle of waste-free recycling taking into account financial benefits. It also requires financial support for R&D projects to develop technologies for creating a new high-value products for an industrial scale. The solution to the problem lies in close collaboration between scientists, developers, and fishers. In our review, we discuss all these parties and hope that we touch upon the interests of all the listed stakeholders in order to unite their efforts in the deep processing of wastes from commercial crab species.

**Author Contributions:** T.P. and E.S. conceived of the presented idea, supervised the findings of this work. E.S. designed the figures, and wrote the manuscript with support from T.P., M.T., M.F., and S.L. S.L. developed the theoretical formalism, performed the analytic calculations and performed the statistical data. M.T., M.F., and S.L. processed the literature data, performed the analysis. All authors discussed the results and contributed to the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** At the time of writing this review, no both human or another living thing were harmed (i.e., did not participate in study).

**Informed Consent Statement:** At the time of writing this review, no both human or another living thing were harmed (i.e., did not participate in study).

**Data Availability Statement:** Most of the data is available on internet platforms, some are available only on paper.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References and Notes**

