Trichoderma Biomass as an Alternative for Removal of Congo Red and Malachite Green Industrial Dyes

: The present study evaluated the removal efﬁciency of two dyes, Congo red (CR) and malachite green (MG), using either fresh or dry fungal biomass of two species of Trichoderma ( T. virens and T. viride ) and activated carbon. After 24 h, the CR removal efﬁciency obtained with fresh biomass was higher than that obtained with activated carbon. For the MG dye, the average removal with activated carbon (99%) was higher than those obtained with dry and fresh biomass of T. viride and T. virens . Experimental results for fresh and dry fungal biomass showed a good correlation with Langmuir isotherms. The adsorption rates of CR and MG by of T. virens and T. viride can be more appropriately described using the pseudo-second-order rate. We found an adsorption capacity of 81.82 mg g − 1 for T. virens with MG dye. Results show that fresh or dry biomass of T. virens can represent a simple and cost-effective alternative for removing industrial dyes such as CR and MG.


Introduction
Synthetic dyes released from textile, cosmetic, paper, and food industries have potentially adverse environmental and health impacts since they not only affect the quality and aesthetic of water but can also be associated with mutagenic, carcinogenic, and allergenic risk [1][2][3][4][5]. Thus, working on new alternatives to remove materials is relevant.
Dyes are complex organic molecules with several recalcitrant aromatic rings classified as anionic (acid), cationic (basic), and non-ionic [6]. They consist of chromophores, which are responsible for giving color, and the auxochromes charge intensifies the color of dyes. Acidic dyes are water-soluble compounds containing one or more anionic groups (salts of sulfuric, carboxylic, phenolic groups). Basic dyes are cationic molecules that have monoazoic, diazoic, and azine compounds.
Among diverse synthetic dyes, Congo red (CR) and malachite green (MG) are two of the most commons due to their low cost, high availability, and efficacy. CR is the sodium salt of benzidinediazo-bis-1-naphthylamine-4-sulfonic acid (C 32 H 22 N 6 Na 2 O 6 S 2 ; molecular weight: 696.66 g mol −1 ) ( Figure 1A). It is an azo acid dye that is very soluble in water, which directly stains cotton in red; it may also turn blue by the presence of mineral acids. CR is also used for staining some products in the wood pulp and paper industry [7,8]. MG, the most common basic dye (N-methylated diaminotriphenylmethane; C 52 H 54 N 4 O 12 ; molecular weight: 927.00 g mol −1 ) ( Figure 1B), has been widely used as a bactericide and fungicide in the fish farming industry, in the coloring of silk, and as a food coloring agent and additive [9,10]. The importance of studying dyes such as CR and MG lies in the fact that they are resistant to natural degradation processes. The aromatic rings provide the dye molecules with chemical stability, so organisms require more energy to mineralize these substances [11,12]. That is to say, since the dye's mineralization process becomes slow, the greater the number of aromatic rings, the longer their persistence in the environment, which could lead to a negative effect on the aquatic flora and fauna [12,13]. Additionally, they are susceptible to being reduced, which results in the formation of aromatic amines that are highly carcinogenic [10,[14][15][16].
To reduce the pollution caused by these two dyes, either chemical, physical, or biological methods have been used [16][17][18]. Some chemical dye removal methods are the advanced oxidation process, ozonation, photochemical, electrochemical destruction, and Fenton reaction. Most of these dye removal methods are effective, quick, and do not generate sludge. However, they could produce undesirable by-products, and in general, they are costly compared to biological and physical dye removal methods. Among conventional physical dye removal methods are adsorption by activated carbon, membrane filtration, ion exchange, electrokinetic flocculation, irradiation, reverse osmosis, and ultrafiltration. Physical methods are commonly used due to their efficiency and simplicity; nevertheless, they are effective for a limited number of dyes, and some of them show high sludge production. On the other hand, biological methods such as adsorption by microbial biomass, algae degradation, fungal cultures, adsorption by living/dead microbial biomass, and enzyme degradation, among others, have gained importance used alone or as a combined method, because they are ecofriendly and do not need the consumption of chemicals. However, these methods deal with living organisms, so they are sometimes considered unstable or unpredictable [19][20][21][22][23].
Among biological treatment with microorganisms, it has been found that the use of living or dead microbial biomass (from bacteria, yeasts, fungi or microalgae) can play a key role in decolorizing the wastewater of different industries without producing toxic substances [24][25][26]. Microbial biomass is a low-cost product obtained as a residue of the fermentation processes in the production of antibiotics, beverages, enzymes, and other metabolites. However, it has been reported that the use of live biomass in the processes of biosorption of dyes presents some disadvantages, such as the need for a continuous supply of nutrients and high sensitivity to the dye toxicity [27]. Therefore, a lot of research has focused on using dead biomass for biosorption processes, since it does not require continuous nutrient supply and is not affected by toxic waste [28]; likewise, its regeneration is simple, can be stored for long periods at room temperature, can be reused for many cycles, and can be easily immobilized on inert supports [26].
The surface properties of bacteria, yeasts, fungi, and algae allow them to interact with different types of contaminants. In particular, in the process of dye biosorption, microbial biomass can use mechanisms such as physical and chemical adsorption, electrostatic interaction, ion exchange, complexation, chelation, and micro-precipitation [29]. Thus, the biosorption process will depend on the dye chemical nature (specie, size, ionic charge), The importance of studying dyes such as CR and MG lies in the fact that they are resistant to natural degradation processes. The aromatic rings provide the dye molecules with chemical stability, so organisms require more energy to mineralize these substances [11,12]. That is to say, since the dye's mineralization process becomes slow, the greater the number of aromatic rings, the longer their persistence in the environment, which could lead to a negative effect on the aquatic flora and fauna [12,13]. Additionally, they are susceptible to being reduced, which results in the formation of aromatic amines that are highly carcinogenic [10,[14][15][16].
To reduce the pollution caused by these two dyes, either chemical, physical, or biological methods have been used [16][17][18]. Some chemical dye removal methods are the advanced oxidation process, ozonation, photochemical, electrochemical destruction, and Fenton reaction. Most of these dye removal methods are effective, quick, and do not generate sludge. However, they could produce undesirable by-products, and in general, they are costly compared to biological and physical dye removal methods. Among conventional physical dye removal methods are adsorption by activated carbon, membrane filtration, ion exchange, electrokinetic flocculation, irradiation, reverse osmosis, and ultra-filtration. Physical methods are commonly used due to their efficiency and simplicity; nevertheless, they are effective for a limited number of dyes, and some of them show high sludge production. On the other hand, biological methods such as adsorption by microbial biomass, algae degradation, fungal cultures, adsorption by living/dead microbial biomass, and enzyme degradation, among others, have gained importance used alone or as a combined method, because they are ecofriendly and do not need the consumption of chemicals. However, these methods deal with living organisms, so they are sometimes considered unstable or unpredictable [19][20][21][22][23].
Among biological treatment with microorganisms, it has been found that the use of living or dead microbial biomass (from bacteria, yeasts, fungi or microalgae) can play a key role in decolorizing the wastewater of different industries without producing toxic substances [24][25][26]. Microbial biomass is a low-cost product obtained as a residue of the fermentation processes in the production of antibiotics, beverages, enzymes, and other metabolites. However, it has been reported that the use of live biomass in the processes of biosorption of dyes presents some disadvantages, such as the need for a continuous supply of nutrients and high sensitivity to the dye toxicity [27]. Therefore, a lot of research has focused on using dead biomass for biosorption processes, since it does not require continuous nutrient supply and is not affected by toxic waste [28]; likewise, its regeneration is simple, can be stored for long periods at room temperature, can be reused for many cycles, and can be easily immobilized on inert supports [26].
The surface properties of bacteria, yeasts, fungi, and algae allow them to interact with different types of contaminants. In particular, in the process of dye biosorption, microbial biomass can use mechanisms such as physical and chemical adsorption, electrostatic interaction, ion exchange, complexation, chelation, and micro-precipitation [29]. Thus, the biosorption process will depend on the dye chemical nature (specie, size, ionic charge), type of biomass, specific surface properties, environmental conditions (pH, temperature, ionic strength), and presence of organic compounds, salts, and competing ions [26].
Fungal adsorption by living or dead biomass is commonly used as biological treatments for dye removal in aqueous media. Fungal biomass contains a large number of functional groups, which gives its cell surface a negative charge that allows the efficient biosorption of dyes [30,31]. However, the process of dye biosorption for fungal biomass can be slow, and the initial pH of the dye solution strongly influences the biosorption [3,32,33].
Filamentous fungi such as Aspergillus niger, Phanerochaete chrysosporium, Rhizopus arrhizus, Cuninghamella elegans, Ganoderma applanatum, and Pleurotus ostreatus, among others, have been assessed as an alternative strategy for removing and degrading dyes under in vitro conditions [34][35][36][37][38][39][40][41]. Concerning the Trichoderma species, previous works have reported that T. harzianum can remove or discolor Rhodamine 6G and Trypan Blue Erioglaucine, and can favor the biosorption of the Orange G dye [42][43][44][45][46]. In addition, researchers have shown the potential of T. viride for the removal of methylene blue [47]. However, more research is needed to determine the true potential of these species for decolorizing polluted waters.
The genus Trichoderma comprises a predominant group of filamentous fungi in terrestrial and aquatic ecosystems widely used in the control of phytopathogenic organisms and industrial processes [48][49][50][51]. The advantages of using species of the genus Trichoderma in the biosorption of dyes are its easy availability, low cost, good mechanical properties, and chemical stability under both alkaline and acid conditions [52]. In this regard, the present study evaluated the capability of either fresh or dry biomass of T. virens and T. viride to remove CR and MG under several dye concentrations. These fungi were selected due to their tolerance to high concentrations of polycyclic aromatic hydrocarbons [53]. In addition, to carry out an objective comparison, under the same conditions, additional absorption experiments were carried out with activated carbon as the sorbent.

Fresh Biomass of T. virens and T. viride
The fungi T. virens (CP1) and T. viride (CP4) were previously isolated from rhizosphere soil of mesquite (Prosopis sp.) in the state of Jalisco, Mexico [54]. The two strains of Trichoderma were reactivated in Petri dishes with potato dextrose ( After six days of incubation, the living mycelium (fresh biomass) was vacuum filtered under sterile conditions. Then, half of the mycelium of each fungal isolate was washed with 50 mL of sterile distilled water and transferred to a 150 mL flask with 50 mL of sterile distilled water. The other half of the fresh fungal biomass was exposed to 0.35 g of HgCl 2 for five days to kill the mycelium and avoid the further synthesis of fungal enzymes [55]. This dead mycelium was subsequently filtrated and washed, as previously described, and placed in 50 mL of sterile distilled water.
Later on, 100 mL of a stock solution of CR or MG (1000 mg L −1 ) were added into each respective treatment (living and dead mycelium) to obtain a final concentration of 50 mg L −1 at pH 6. The treatments were incubated at 28 ± 2 • C at 150 rpm for eight days, sampling every 24 h; then, 300 µL of each treatment were transferred to a microplate and analyzed in a multi-modal microplate reader (Synergy 2 SL, Biotek ® , Winooski, VT, USA) to 541 nm (CR) and 619 nm (MG), respectively. The amount of dye removal per unit of fungal biomass was calculated using the following equation: where C i and C f represent the initial and final (after adsorption) dye concentrations, respectively.

Activated Carbon and Dry Biomass of T. virens and T. viride
First, 500 mL of the mineral medium previously described were added to 1000 mL Erlenmeyer flasks and autoclaved for 18 min at 120 • C. Then, 10 mL of a spore suspension (10 6 spores mL −1 ) of each fungus were subsequently added to each culture. Fungal cultures were incubated at room temperature and pump oxygenated for six days. After incubation, the fungal mycelium was vacuum filtered, dried at 70 ± 2 • C for 96 h, weighed, and powdered in a mortar.
Removal experiments were carried out by stirring 100 mg of the respective adsorbent material [activated carbon Sigma Aldrich ® (State of Mexico, Mexico) and fungal dry biomass] with 1 mL of CR or MG at the following concentrations: 0, 15, 30, 45, 60, 75, and 90 mg L −1 at pH 6, and incubated at 25 • C and 150 rpm (TS-100, Biosan ® , Riga, Latvia). The concentration of each dye was spectrophotometrically estimated by taking absorbance readings at 541 nm for CR and 619 for MG, using 96-well microplates (Synergy 2 SL, Biotek ® , Winooski, VT, USA). Removal capacity was determined using Equation (1).

Equilibrium Studies
The equilibrium relationship between the amount of substance adsorbed at constant temperature (q e ) and its equilibrium solution concentration (C e ) is known as adsorption isotherm. The equilibrium adsorption data were analyzed using Langmuir and Freundlich models. The linearized forms of the Langmuir and Freundlich isotherms are given by: where q max (mg g −1 ) is the maximum value of q e that can be reached as C e (mg g −1 ) is increased, K L is the affinity coefficient (L mg −1 ), and K F and n represent the Freundlich coefficients (n, dimensionless; K F , mg g −1 ).

Kinetic Studies
The adsorption kinetics was analyzed with models of pseudo-first and pseudo-second order. The pseudo-first-order model, in its linear form is described by Lagergren: where q e is the adsorption capacity at equilibrium (mg g −1 ), q t is the amount of the adsorbate adsorbed by time t (mg g −1 ), and k 1 is the pseudo-first-order constant (min −1 ). The linear form of the pseudo-second-order kinetic model is described by Ho: where k 2 is the pseudo-second-order constant (g mg −1 min −1 ).

Statistic Analysis
The experiment for the removal of CR and MG by fresh biomass (with or without HgCl 2 treatment) of T. virens and T. viride was set in a completely randomized experimental design, establishing a 2 × 2 × 2 factorial experiment (two strains of Trichoderma, two dyes, and two conditions of biomass), including eight treatments and three replicates each. Analysis of variance and mean comparison test (Tukey, α = 0.05) were performed using the SAS statistical program.
It has been reported that the biomass of some other filamentous fungi shows a higher capacity for removing CR and MG (Table 1). However, T. viride and T. virens have the advantage of growing rapidly in conventional media and agri-food residues and produce, at low-cost, large quantities of biomass, compared to some strains of Aspergillus niger, Polyporus elegans, T. versicolor, and Pleurotus ostreatus [62][63][64].
It is worth to note that in this research, HgCl2 was used to kill mycelium and prevent the synthesis of fungal enzymes, according to the methodology of Wunch et al. [55]. How- The living mycelium in the two species of Trichoderma had slightly lower removal capability of CR when compared to that described for Trametes versicolor (100%) and Thelephora sp. (97%); however, the concentration of CR used in the present study (50 mg L −1 ) is higher than those reported for T. versicolor or Thelephora with 31 mg L −1 and 35 mg L −1 , respectively [57] (Table 1). In contrast, when the CR concentration was 50 mg L −1 , the removal observed for T. versicolor, 82%, for Aspergillus niger, 9%, for A. oryzae, 52%, for Penicillium chrysogenum, 10%, for Cladosporium rubrum, 10%, and Pleurotus ostreatus, 12% [58,59], were lower than that obtained in the present study. The removal percentages of CR by the dead mycelium of T. virens (94%) and T. viride (95%) are higher than those described for the mycelium of T. versicolor under three conditions: (a) autoclaved (90%), (b) acidic (49%), and (c) alkaline (42%) [58] (Table 1).
It has been reported that the biomass of some other filamentous fungi shows a higher capacity for removing CR and MG (Table 1). However, T. viride and T. virens have the advantage of growing rapidly in conventional media and agri-food residues and produce, at low-cost, large quantities of biomass, compared to some strains of Aspergillus niger, Polyporus elegans, T. versicolor, and Pleurotus ostreatus [62][63][64].
It is worth to note that in this research, HgCl 2 was used to kill mycelium and prevent the synthesis of fungal enzymes, according to the methodology of Wunch et al. [55]. However, its use is not recommended because of its environmental implications. Another alternative to kill mycelium in an environmentally friendly way is autoclaving [58]. In addition, autoclaving mycelium has been found to increase the adsorption of fungal biomass [26].
To evaluate the adsorption rate, the data obtained from kinetic experiments were fitted using pseudo-first (Equation (4)) and pseudo-second-order (Equation (5)) reaction rate models. The results indicated that the adsorption rates of CR and MG by T. virens and T. viride could be more appropriately described using the pseudo-second-order rate model (correlation coefficient R 2 = 0.99). Figure 3A (CR) and 3B (MG) show plots of experimental data fitted to the pseudo-second-order rate model. Table 2 gives the parameters determined from the linear regression plots (t/q t vs. t) using the pseudo-second-order model and the experimental values of q e (q e,exp ) for both dyes. ever, its use is not recommended because of its environmental implications. Another alternative to kill mycelium in an environmentally friendly way is autoclaving [58]. In addition, autoclaving mycelium has been found to increase the adsorption of fungal biomass [26]. To evaluate the adsorption rate, the data obtained from kinetic experiments were fitted using pseudo-first (Equation (4)) and pseudo-second-order (Equation (5)) reaction rate models. The results indicated that the adsorption rates of CR and MG by T. virens and T. viride could be more appropriately described using the pseudo-second-order rate model (correlation coefficient R 2 = 0.99). Figure 3A (CR) and 3B (MG) show plots of experimental data fitted to the pseudo-second-order rate model. Table 2 gives the parameters determined from the linear regression plots (t/qt vs. t) using the pseudo-second-order model and the experimental values of qe (qe,exp) for both dyes.    Figure 4 shows removal efficiencies for CR ( Figure 4A) and MG ( Figure 4B), for the more representative dye concentrations, as a function of stirring times ranging from 1.5 to 48 h. It is important to note that removal by activated carbon is very fast for both dyes and remained almost unchanged during the experimentation time, indicating that it reaches the equilibrium faster than the mycelium of both Trichoderma isolates. As it can be seen, during the first 24 h, the activated carbon showed the highest CR removal capacity. In absorption processes, activated carbon is a highly effective and versatile material. However, both chemical and thermal regeneration of the used carbon produces effluent, is expensive, and is impractical on a large scale.  On the other hand, after 24 h, the dry biomass of both Trichoderma species showed higher CR removal capacity than activated carbon ( Figure 4A). In addition, the CR removal obtained by the dry biomass of T. virens (88%) was higher than that reported for the fungus Aspergillus niger (72%) [73]. In contrast, MG removal by the dry biomass of both fungi was lower than that observed by activated carbon during the first 24 h, although, at 48 h, the removal of MG by the dry fungal biomass was similar to that observed for activated carbon ( Figure 4B).

Removal of CR and MG by Activated Carbon and Dry Biomass of T. virens and T. viride
Equilibrium adsorptions data of CR and MG onto activated carbon and dry biomass of the two Trichoderma species were analyzed using the Freundlich (Equation (2)) and Langmuir (Equation (3)) adsorption isotherms. The isothermal plots were found to be linear, and the linear correlation coefficients were slightly higher for the Langmuir model ( Figure 5); all the calculated isotherm model parameters are given in Table 2. On the other hand, after 24 h, the dry biomass of both Trichoderma species showed higher CR removal capacity than activated carbon ( Figure 4A). In addition, the CR removal obtained by the dry biomass of T. virens (88%) was higher than that reported for the fungus Aspergillus niger (72%) [73]. In contrast, MG removal by the dry biomass of both fungi was lower than that observed by activated carbon during the first 24 h, although, at 48 h, the removal of MG by the dry fungal biomass was similar to that observed for activated carbon ( Figure 4B).
Equilibrium adsorptions data of CR and MG onto activated carbon and dry biomass of the two Trichoderma species were analyzed using the Freundlich (Equation (2)) and Langmuir (Equation (3)) adsorption isotherms. The isothermal plots were found to be linear, and the linear correlation coefficients were slightly higher for the Langmuir model ( Figure 5); all the calculated isotherm model parameters are given in Table 2. Additionally, we calculate the equilibrium parameter, RL, which represent an important characteristic in the Langmuir model and is defined by where C0 stands for the initial concentration of dye. The value of RL indicates favorable absorption if 0 < RL <1. In addition, RL = 0 denotes irreversible absorption, RL = 1 indicates linear absorption, and RL >1 reveals unfavorable absorption [74]. Then, higher correlation coefficients for the Langmuir model and RL values between 0 and 1 (last column in Table  3) suggest that the adsorption of CR and MG by activated carbon and dry biomass of T. virens and T. viride could be well described by the Lamgmuir model.   (2)) and Langmuir (Equation (3)) adsorption isotherm for Congo red (row A) and malachite green (row B).
Additionally, we calculate the equilibrium parameter, R L , which represent an important characteristic in the Langmuir model and is defined by where C 0 stands for the initial concentration of dye. The value of R L indicates favorable absorption if 0 < R L < 1. In addition, R L = 0 denotes irreversible absorption, R L = 1 indicates linear absorption, and R L >1 reveals unfavorable absorption [74]. Then, higher correlation coefficients for the Langmuir model and R L values between 0 and 1 (last column in Table 3) suggest that the adsorption of CR and MG by activated carbon and dry biomass of T. virens and T. viride could be well described by the Lamgmuir model. Parameters found through the Freundlich model could give information about the heterogeneous nature of adsorption (n > 1). That is the case of CR adsorption by activated carbon and T. virens, and MG adsorption by T. virens. However, linear correlation coefficients (R 2 ) are higher for the Langmuir model than for the Freundlich model. For the Langmuir isotherm, the saturation capacity of the monolayer in the activated carbon (10.7) is greater than that for T. virens (5.6) and T. viride (2.76), for CR; whereas for the MG dye, T. virens (81.8) had higher saturation capacity of the monolayer when compared to T. viride and activated carbon. The constant K L for CR dye had similar values in all three adsorbent materials, whereas for MG, the K L was greater for activated carbon (Table 3).
Reviewing the reports for the monolayer saturation capacity of other organic and inorganic adsorbents used for the removal of CR dye (Table 4), it was found that most of the materials showed a higher saturation capacity of the monolayer than that obtained for the two species of Trichoderma reported in this study: straw carbon (403.7), grapefruit peel carbon (169.5), ground nut shells charcoal (117.6), bamboo dust carbon (101.9), Ca-bentonite (85.3), hen feather (73.8), and cassava residue (59.2), among others. Nevertheless, the saturation capacity was higher than that reported for other adsorbents such as laboratory-grade activated carbon and commercial-grade activated carbon, with 1.88 and 0.64, respectively [75][76][77]. On the other hand, for MG, few materials showed higher saturation capacity of the monolayer than T. virens (Table 4) . Finally, it is worth noting the performance of the Ackee apple seed-bentonite composite, which showed the highest saturation capacity for both CR (1439.9) and MG (706.7). Thus, an alternative to improve the adsorption capacity of dry biomass of T. virens is to apply an acid or alkaline pre-treatment or combine it with other materials or biomasses from other fungi or microorganisms.
On the other hand, the kinetics studies reveal that adsorption rates of CR and MG by activated carbon and dry biomass of T. virens and T. viride are adequately described by the pseudo-second-order rate model ( Figure 6). Table 5 shows the values obtained from the linear regression plots (t/q t vs. t) using the pseudo-second-order model, for the different concentrations of both dyes, finding that q e tends to increase along with C e in the three adsorbents tested in this study; the highest values of q e were obtained with activated carbon. On the other hand, the kinetics studies reveal that adsorption rates of CR and MG by activated carbon and dry biomass of T. virens and T. viride are adequately described by the pseudo-second-order rate model ( Figure 6). Table 5 shows the values obtained from the linear regression plots (t/qt vs. t) using the pseudo-second-order model, for the different concentrations of both dyes, finding that qe tends to increase along with Ce in the three adsorbents tested in this study; the highest values of qe were obtained with activated carbon.

Conclusions
The removal of dyes depends on physicochemical factors such as the concentration and size of the dye molecule, ionic charge, pH, and temperature. In this research, it was observed that the genotype and the preparation of the fungal biomass are relevant factors when looking for high removal percentages. In that sense, it was found that the removal of CR by the fresh biomass of both species of Trichoderma studied was very similar. For MG, the highest percentage of removal was obtained with the live and dead biomass of T. virens.
In the same way, the present study showed that after 24 h of contact time, fresh and dry biomass of T. virens and T. viride had a higher CR removal capacity than activated carbon. Additionally, T. virens showed a higher saturation capacity of the monolayer than T. viride and activated carbon for MG removal. Thus, either fresh or dry biomass of T. virens can represent a simple and cost-effective alternative for removing industrial dyes like CR and MG.