**2. Materials and Methods**

**1.6. Marine fungi**

OH

Cl Cl

Cl

OH

Cl Cl

OH

CO2 + H2O

Cl

202 Advances in Bioremediation of Wastewater and Polluted Soil

Cl

Cl

Cl

TCHQ tetrachloro-1,4-

CH3O

OCH3

Cl

OCH3

Cl Cl

OCH3

dimethoxybenzene

OCH3

O H 3,4-dimethoxy benzaldehyde

Cl

Cl

OCH3

OCH3

2-methoxyphenol

OCH3

O OH

3-hydroxy-4-methoxy benzoic acid

HO

HO

OCH3

OCH3

O H

3-hydroxy-4-methoxy benzaldehyde

2-chloro-1,4 dimethoxybenzene

Cl

pentachloro anisole

Cl Cl

Cl

Cl

The marine environment covers more than three quarters of the Earth's surface and is a promising source of new enzymes [104]. These enzymes show great potential for use in biocatalytic reactions by possessing unique characteristics related to the marine environment. In recent years, a wide variety of enzymes and microorganisms with specific activities have been isolated from marine environments [105] and have been extensively studied, particularly

**Figure 4.** Degradation of PCP by the fungus *Anthracophyllum discolor* (Source: modified from Rubilar et al. [101]).

HO

The words "marine fungi" are not derived from a taxonomic class and they are not classified by their physiological characteristics. These fungi considered as an ecological group, and the most suitable definition was proposed by Kohlmeyer and Kohlmeyer [107]: "Mandatory marine fungi are those that grow and sporulate exclusively in a marine or estuarine habitat; facultative marine fungi are those from freshwater or terrestrial water environments and are able to grow and even sporulate in the marine environment " [108]. In the marine environment,

proteases, carbohydrases, oxidoreductases, peroxidases [106].

#### **2.1. Isolation of fungi strains**

Marine-derived fungi were isolated from the ascidian *Didemnum ligulum* according to the method described by Kossuga et al. [124]. The ascidian samples were collected in São Sebastião, South Atlantic Ocean, in September 2005 at the northern coast of São Paulo state, Brazil, by Prof. Roberto G.S. Berlinck (IQSC-USP, Brazil). After the isolation and purification of the strains, the marine-derived fungi were deposited in the microbiology laboratory of the Department of Ecology and Aquatic Microbiology supervised by Mirna H.R. Seleghim (UFSCar, Brazil). They were preserved by two techniques: in distilled water according to Castellani [125] and in inclined tubes containing agar, both stored under refrigeration. The strains were reactivated for the experiments by streaking or aseptic transfer of mycelial discs to solid culture media (3% malt).

In the laboratory, samples collected from the ascidian were subjected to surface sterilization by successive washes with 0.001 g.L–1 solution of HgCl2 in 5% ethanol for 1 minute, followed by 3 washes with sterile sea water [126]. Then, portions of about 1 cm2 were taken from the inside of the ascidian with a sterile scalpel. These fragments were inoculated in Petri dishes containing agar medium with artificial sea water (ASW - Artificial Sea Water) and the broadspectrum antibiotic rifampicin (0.3%) to inhibit bacterial growth [127]. Plates were incubated for 7 d at 25° C. Eight culture media were prepared (Table 3) in order to expand the possibilities of obtaining different strains that may be associated with the ascidian *D. ligulum*.


**Table 3.** Culture media composition for isolation of marine-derived fungi from *Didemnun ligulum* [124].

## **2.2. Purification**

strains, the marine-derived fungi were deposited in the microbiology laboratory of the Department of Ecology and Aquatic Microbiology supervised by Mirna H.R. Seleghim (UFSCar, Brazil). They were preserved by two techniques: in distilled water according to Castellani [125] and in inclined tubes containing agar, both stored under refrigeration. The strains were reactivated for the experiments by streaking or aseptic transfer of mycelial discs

In the laboratory, samples collected from the ascidian were subjected to surface sterilization by successive washes with 0.001 g.L–1 solution of HgCl2 in 5% ethanol for 1 minute, followed by 3 washes with sterile sea water [126]. Then, portions of about 1 cm2 were taken from the inside of the ascidian with a sterile scalpel. These fragments were inoculated in Petri dishes containing agar medium with artificial sea water (ASW - Artificial Sea Water) and the broadspectrum antibiotic rifampicin (0.3%) to inhibit bacterial growth [127]. Plates were incubated for 7 d at 25° C. Eight culture media were prepared (Table 3) in order to expand the possibilities

of obtaining different strains that may be associated with the ascidian *D. ligulum*.

2% Malt Extract Agar (MA2%) Malt extract (20 g L–1), agar (15 g L–1) in artificial seawater 3% Malt Extract Agar (MA3%) Malt extract (30 g L–1), mycological peptone (5 g L–1) and agar

(15 g L–1) in artificial seawater

(15 g L–1) in artificial seawater

seawater to 1 L with agar (15 g L–1)

(5 g L–1), agar (20 g L–1) in artificial seawater

12 h, filtered, and then the supernatant was diluted with artificial

filtered, and then diluted with artificial seawater to 1 L with agar

potassium phosphate (1.0 g L–1 KH2PO4), magnesium sulfate heptahydrate (0, 5 g L–1, MgSO4.7H2O), 0.01 g of iron(II) sulphate heptahydrate (0.01 g L–1 FeSO4.7H2O), agar (15 g L–1) in artificial

Glucose agar, Peptone, and Yeast extract (GPY) Glucose (1 g L–1), soy peptone (0.5 g L–1), yeast extract (0.1 g L–1), agar

Potato Carrot Agar (PCA) Cooked and mashed potatoes (20 g L–1), cooked and mashed carrots

Corn Meal Agar (CMA) Maize flour (42 g L–1) stirred in 500 mL of distilled water at 60°C for

Oat Meal Agar (OMA) Rolled oats (30 g) were boiled in 500 mL of distilled water for 1 h,

Tubaki Agar (TA) Glucose (30 g L–1), yeast extract (0.5 g L–1), peptone (1.0 g L–1), dibasic

Cellulose Agar (CA) Cellulose (10 g L–1), yeast extract (1 g L–1), agar (15 g L–1) in artificial

(20 g L–1)

seawater

seawater

**Table 3.** Culture media composition for isolation of marine-derived fungi from *Didemnun ligulum* [124].

to solid culture media (3% malt).

204 Advances in Bioremediation of Wastewater and Polluted Soil

**Culture media Composition**

The Petri dishes with different culture media containing the filamentous fungi strains were examined periodically. The isolated strains were subjected to successive inoculations to obtain pure cultures. Initially, the pure cultures were described by morphological method and coded as DL. The DL code was related to the organism from which the strains were isolated, the ascidian *Didemnum ligulum*, and the abbreviation for the culture medium used in the isolation. Eight different culture media for strain isolation were used; however, fungi growth was not observed in the cellulose agar and Tubaki agar media.

The 15 isolated strains were coded as; DL5A, DL6A, DL11A (oatmeal agar medium), DL2B, DL5B (potato carrot agar medium), DL1F, DL2F, (corn meal agar medium), DL5G, (glucose agar, peptone, and yeast extract culture medium), DL3M2, (2% malt extract agar medium), DL1M3, DL4M3, DL6M3, DL7M3, DL8M3, and DL9M3 (3% malt extract agar medium). The detailed methodology for the isolation and purification were described by Kossuga et al. [124]. The procedures were performed at the Department of Ecology and Evolutionary Biology at UFSCar, São Carlos, Brazil.

#### **2.3. Identification of strains by molecular biology**

The 15 fungal strains were characterized and identified by techniques based on the molecular identification of genes rRNA, ITS1 and ITS4. These analyzes were carried out under the supervision of Prof. Dr. Suzan Pantaroto de Vasconcellos at the Federal University of São Paulo (UNIFESP), Campus Diadema.

The isolates were grown on yeast extract sucrose agar (YES) (10 g yeast extract, 75 g sucrose, 10 g agar, and 500 mL distilled water). Then, DNA was extracted with the PrepMan Ultra sample preparation reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The DNA concentration and purity (relative to proteins and salts) were determined by optical density at 260 nm (OD260) and ratios of OD260/280 and OD260/230, respectively. The internal transcribed spacer (ITS) region of rDNA were amplified with primer pairs and ITS1/ITS4 using the protocol described by Gonçalves et al. (2012). The reactions were performed with PCR master mix (Promega, Madison, WI, USA) according to the manufacturer's instructions. After amplification, the fragments were sequenced following the protocol provided with the BigDye reagent kit (Applied Biosystems, Foster City, CA, USA) in an ABI 3130 (Applied Biosystems, Foster City, CA, USA) automatic sequencer. PCR products were sequenced with the same primers used for amplification. Contig assembly and editing were performed with Sequencher DNA sequence assembly software 4.1.4 (Gene Codes Corporation, Ann Arbor, MI, USA). Successful assembly of the contigs required a minimum match percentage of 85 and a minimum overlap of 20.

Complete ITS consensus sequences were used to conduct BLAST search analysis for species identification from the NCBI genomic database (http://blast.ncbi.nlm.nih.gov/).

For all regions analyzed by BLAST search, the sequences that were presented with high identity (99%), queries, and E values of e10–5 were considered for the final species identification using the sequencing method.

#### **2.4. Growth of fungi strains in solid medium**

The strains of marine-derived fungi were cultivated on Petri dishes containing 3% malt solid medium using artificial sea water with the following composition: malt extract (30.0 g L–1), soy peptone (3.0 g L–1), and agar (20.0 g L–1). The pH was adjusted to 8 with KOH solution (0.1 mol L–1), similar to the pH of the marine environment [124]. Artificial seawater composition was: CaCl2.2H2O (1.36 g L–1), MgCl2.6H2O (9.68 g L–1), KCl (0.61 g L–1), NaCl (30.0 g L–1), Na2HPO4 (0.014 mg L–1), Na2SO4 (3.47 g L–1), NaHCO3 (0.17 g L–1), KBr (0.10 g L–1), SrCl2.6H2O (0.04 g L– 1 ), and H3BO3 (0.03 g L–1).

#### **2.5. Selection of fungal strains resistant to PCP in solid culture medium**

Fifteen fungal strains were grown in Petri dishes and inoculated in solid medium containing 3% malt extract medium without PCP (control) and with different concentrations of the organochlorine pesticide; 10, 25, 30, 40, and 50 mg L–1 per plate (98%, analytical standard commercially obtained from Sigma-Aldrich, Brazil). The experiments were prepared in triplicate. Ethyl acetate was used as solvent to prepare the stock solution of the pesticide in the proportion of 5.0 mg of PCP / 100 μL of ethyl acetate.

The culture media were sterilized in an autoclave at 121 °C for 20 minutes, cooled to about 40-50 °C, and then the pesticide stock solution was added, according to the desired concen‐ tration. The mixture was homogenized and then added in Petri dishes. The inoculation of fungi was made by transferring the mycelium of pure cultures precultivated in 3% malt medium after 5 d of growth by a platinum needle insertion point into the plate center. The plates were incubated at 32 °C (B.O.D. 411D, Nova Ética) and the radial growth of the fungus were observed for 21 d. The diameter of the colony formed was measured at 7 d intervals, as performed by Birolli et al. [128]. The strain that showed the highest radial growth was selected for the PCP biodegradation in a liquid medium. The experiments were performed in triplicates.

## **3. Results and Discussion**

The aim of this chapter was the isolation and selection of marine-derived fungi with potential for PCP biodegradation. So the PCP biodegradation details will not be discussed because they already were published. Figure 5 shows the 15 fungi strains isolated from *Didemnum ligulum* cultivated in 3% malt extract medium in absence of pesticide.

The isolated fungi were identified by molecular biology and exhibited a variety of genera and species illustrating the fungi diversity in marine environment (Table 4): *T. harzianum* CBMAI 1677 was deposited in the Brazilian Collection of Environmental and Industrial Microorgan‐ isms (CBMAI - http://webdrm.cpqba.unicamp.br/cbmai/, WDCM 823).


**Table 4.** The codification and identification of the strains employed in this study.

**2.4. Growth of fungi strains in solid medium**

206 Advances in Bioremediation of Wastewater and Polluted Soil

proportion of 5.0 mg of PCP / 100 μL of ethyl acetate.

cultivated in 3% malt extract medium in absence of pesticide.

isms (CBMAI - http://webdrm.cpqba.unicamp.br/cbmai/, WDCM 823).

1

), and H3BO3 (0.03 g L–1).

**3. Results and Discussion**

The strains of marine-derived fungi were cultivated on Petri dishes containing 3% malt solid medium using artificial sea water with the following composition: malt extract (30.0 g L–1), soy peptone (3.0 g L–1), and agar (20.0 g L–1). The pH was adjusted to 8 with KOH solution (0.1 mol L–1), similar to the pH of the marine environment [124]. Artificial seawater composition was: CaCl2.2H2O (1.36 g L–1), MgCl2.6H2O (9.68 g L–1), KCl (0.61 g L–1), NaCl (30.0 g L–1), Na2HPO4 (0.014 mg L–1), Na2SO4 (3.47 g L–1), NaHCO3 (0.17 g L–1), KBr (0.10 g L–1), SrCl2.6H2O (0.04 g L–

Fifteen fungal strains were grown in Petri dishes and inoculated in solid medium containing 3% malt extract medium without PCP (control) and with different concentrations of the organochlorine pesticide; 10, 25, 30, 40, and 50 mg L–1 per plate (98%, analytical standard commercially obtained from Sigma-Aldrich, Brazil). The experiments were prepared in triplicate. Ethyl acetate was used as solvent to prepare the stock solution of the pesticide in the

The culture media were sterilized in an autoclave at 121 °C for 20 minutes, cooled to about 40-50 °C, and then the pesticide stock solution was added, according to the desired concen‐ tration. The mixture was homogenized and then added in Petri dishes. The inoculation of fungi was made by transferring the mycelium of pure cultures precultivated in 3% malt medium after 5 d of growth by a platinum needle insertion point into the plate center. The plates were incubated at 32 °C (B.O.D. 411D, Nova Ética) and the radial growth of the fungus were observed for 21 d. The diameter of the colony formed was measured at 7 d intervals, as performed by Birolli et al. [128]. The strain that showed the highest radial growth was selected for the PCP biodegradation in a liquid medium. The experiments were performed in triplicates.

The aim of this chapter was the isolation and selection of marine-derived fungi with potential for PCP biodegradation. So the PCP biodegradation details will not be discussed because they already were published. Figure 5 shows the 15 fungi strains isolated from *Didemnum ligulum*

The isolated fungi were identified by molecular biology and exhibited a variety of genera and species illustrating the fungi diversity in marine environment (Table 4): *T. harzianum* CBMAI 1677 was deposited in the Brazilian Collection of Environmental and Industrial Microorgan‐

**2.5. Selection of fungal strains resistant to PCP in solid culture medium**

For the evaluation of the fungi inhibition caused by the presence of the xenobiotic compound, radial growth experiments were performed. The marine-derived fungi were cultivated in various concentrations of PCP (10, 25, 30, 40, and 50 mg.L–1 per plate). The inoculation was carried out by a central insertion point using an inoculation needle. After incubation, the colonies' diameters were measured at 7, 14, and 21 d. The results are summarized in Tables 4-6.

All marine-derived fungi showed excellent growth after 7 d of cultivation in solid culture medium (3% mat extract agar) without PCP. The results showed that 3% malt extract medium was suitable for growth of marine-derived fungi as suggested by Kjer et al. [130]. After 21 d of incubation, 60% of the strains have grown throughout the plate surface, reaching 8.0 cm of colony diameter (diameter of the employed Petri dish). The cultivation of the fungus in the absence of PCP was important to assess the development of the pure cultures isolated from the sponge *D. ligulum*.

In the presence of the organochlorine pesticide, the strains coded as DL1M3, DL4M3, DL6M3, DL7M3, DL8M3, and DL9M3 failed to grow in any of the plates containing PCP, showing low

medium. **Figure 5.** Colonies of marine-derived fungi isolated from the ascidian *Didemnum ligulum* grown in 3% malt extract me‐ dium.

Figure 5. Colonies of marine-derived fungi isolated from the ascidian *Didemnum ligulum* grown in 3% malt extract

6

resistance and adaptation to the organochlorine presence, and thus suggested low potential for biodegradation. It is noteworthy that these microorganisms were isolated from environ‐ ments without PCP contamination; therefore its presence caused growth inhibition because this is a very toxic compound for living organisms and these strains were not adapted to its effects on their metabolism.


a Standard deviation: minimum (0.07 cm) and maximum (0.4 cm).


6

*Pleosporales* sp. DL1F

208 Advances in Bioremediation of Wastewater and Polluted Soil

*Penicillium citrinum*  DL4M3

*Didymella phacae* DL7M3

*Cladosporium cladosporioides* DL5B

*Cladosporium cladosporioides* DL2F

medium.

dium.

*Stagonosporopsis cucurbitacearum* DL1M3

*Aspergillus versicolor* DL5A

> *Phoma* sp. DL8M3

*Fusarium fujikuroi* DL11A

*Mycosphaerella crystallina* DL6M3

**Figure 5.** Colonies of marine-derived fungi isolated from the ascidian *Didemnum ligulum* grown in 3% malt extract me‐

Figure 5. Colonies of marine-derived fungi isolated from the ascidian *Didemnum ligulum* grown in 3% malt extract

*Trichoderma harzianum* DL2B

*Cladosporium cladosporioides* DL5G

*Aspergillus versicolor* DL6A

> *Cladosporium cladosporioides* DL3M2

> > All experiments in plates were performed in triplicate.

**Table 5.** Average diameter of fungi colonies isolated from the ascidian *D. ligulum* after 7 d of growth (32° C, 3% malt extract medium) in the presence and absence of PCP.


a Standard deviation: minimum (0.0 cm) and maximum (0.2 cm).


All experiments in plates were performed in triplicate.

**Table 6.** Average diameter of fungi colonies isolated from the ascidian *D. Ligulum* after 14 d of growth (32° C, 3% malt extract medium) in the presence and absence of PCP.

As shown in Tables 5-7, some strains did not grow in the presence of PCP. In addition, the strains capable of growth in the employed conditions showed that the more concentrated the PCP, the less growth presented in the culture medium. These results indicated that PCP causes a toxic effect on these microorganisms. However, the fact that the majority of the strains subjected to this experiment grew, at least, in one of the tested concentrations indicates that


a Standard deviation: minimum (0.1 cm) and maximum (0.5 cm).


**Colony diameter (cm)a**

**25 (mg.mL–1 )**

3.6 - - - - -

4.2 - - - - -

3.1 - - - - -

1.5 - - - - -

5.4 - - - - -

2.6 0.4 - - - -

6.7 3.6 1.7 0.1 - -

4.8 3.0 1.6 0.3 - -

8.0 4.9 1.8 1.6 - -

8.0 6.6 5.9 2.7 1.4 2.1

**10 (mg.mL–1 )**

*S. cucurbitacearum* DL1M3 6.6 - - - - -

*C. cladosporioides* DL2F 4.3 1.2 - - - - *C. cladosporioides* DL3M2 4.3 1.3 - - - - *C. cladosporioides* DL5B 3.1 1.5 - - - - *C. cladosporioides* DL5G 4.8 3.4 - - - -

**Table 6.** Average diameter of fungi colonies isolated from the ascidian *D. Ligulum* after 14 d of growth (32° C, 3% malt

As shown in Tables 5-7, some strains did not grow in the presence of PCP. In addition, the strains capable of growth in the employed conditions showed that the more concentrated the PCP, the less growth presented in the culture medium. These results indicated that PCP causes a toxic effect on these microorganisms. However, the fact that the majority of the strains subjected to this experiment grew, at least, in one of the tested concentrations indicates that

**Concentration of PCP in Petri dishes with PCP**

**30 (mg.mL–1 )**

**40 (mg.mL–1 )**

**50 (mg.mL–1 )**

**Strains Petri dishes**

210 Advances in Bioremediation of Wastewater and Polluted Soil

*P. citrinum* DL4M3

*M. crystallina* DL6M3

*D. phacae* DL7M3

*Phoma* sp. DL8M3

Not identified DL9M3

*Pleosporales* sp. DL1F

*A. versicolor* DL6A

*A. versicolor* DL5A

*F. fujikuroi* DL11A

*T. harzianum* CBMAI 1677


a

**without PCP**

Standard deviation: minimum (0.0 cm) and maximum (0.2 cm).

All experiments in plates were performed in triplicate.

extract medium) in the presence and absence of PCP.

All experiments in plates were performed in triplicate.

**Table 7.** Average diameter of fungi colonies isolated from the ascidian *D. Ligulum* after 21 d of growth (32 C, 3% malt extract medium) in the presence and absence of PCP.

the toxic effect exerted by the compound was not enough to prevent fungal resistance and consequently, biodegradation potential.

According to Bonugli-Santos et al. [129] and Ortega et al. [116], marine-derived microorgan‐ isms tend to be resistant when subjected to adverse conditions and can be used in bioreme‐ diation techniques because they have enzymes adapted to complex environments such as those with extreme pressure, salinity, and temperature variations. They are able to develop impor‐ tant metabolic and physiological activities, for example, degradative potential of organochlor‐ ine pesticides.

The best adapted strain to the presence of PCP were by DL6A, DL5A, DL11A, and DL2B strains, which were capable to grow at concentrations above 10 mg L–1. The colony diameter of the strains DL6A and DL5A increased in the concentrations of 10, 20, and 30 mg L–1, but the sizes were inferiorincomparisonwithDL11AandDL2Bstrains inthe same concentrations (Figure6).

*Aspergillus versicolor* DL6A

Figure 6. Growth of marine-derived fungi (DL6A, DL5A, DL11A, DL2B) in 3% malt extract agar containing different concentrations of PCP after 21 d at 32° C. The plate numbers of 1, 2, 3, 4, and 5, respectively, correspond to 10, 25, 30, 40, and 50 mg L–1 of PCP. **Figure 6.** Growth of marine-derived fungi (DL6A, DL5A, DL11A, DL2B) in 3% malt extract agar containing different concentrations of PCP after 21 d at 32° C. The plate numbers of 1, 2, 3, 4, and 5, respectively, correspond to 10, 25, 30, 40, and 50 mg L–1 of PCP.

Earlier studies have shown that adaptation experiment with fungi in solid culture medium is a simple and important methodology to screen microorganisms for pesticide biodegradation [133]. After the adaptation experiments with the 15 isolated strains, The most part of microorganisms show increasing growth inhibition in increasing xenobiotics concentrations, especially on those with high toxicity. However, the strain DL2B, which showed the best results in the solid media experiment also grew well at the highest pesticide concentration (50 mg L–1). Thus, this fungus showed resistance to toxicity, adaptive capacity, and biodegradation potential for PCP, even at high concentrations. Creswell and Curl [131] achieved similar results assessing the growth of the fungus *Trichoderma harzianum* in the presence of herbicides such as prometryn, norflurazon, and ciazine. In this work, the fungal growth was significantly increased at the highest dose of the herbicide norflurazon. According to Tomasini et al. [132] fungi need a period of adaptation in high toxicity conditions and, if they were resistant, in the final period of cultivation they tend to grow more. If a group of microorganisms can proliferate efficiently in environments with high concentrations of certain pollutants, it is an indication that these microorganisms have a metabolism adapted to the presence of these contaminants [62]. The increased growth in the presence of the xenobiotic can occur because of its use as nutrient, especially carbon source.

Earlier studies have shown that adaptation experiment with fungi in solid culture medium is a simple and important methodology to screen microorganisms for pesticide biodegradation [133]. After the adaptation experiments with the 15 isolated strains, *Trichoderma harzianum* DL2B (CBMAI 1677) was selected for studies of biotransformation and biodegradation of PCP. In a later study, Vacondio et al. [134] observed that after 7 d of incubation with 20 mg L–1 of PCP in liquid medium, it was no longer detected in the presence of PCP in the samples, showing the biodegradation of the pesticide by *Trichoderma harzianum* CBMAI 1677. In addition, the metabolites pentachloroanisole (PCA) and 2,3,4,6-tetrachloroanisole (2,3,4,6- TeCA) were identified. *T. harzianum* was also able to biodegrade PCA and 2,3,4,6-TeCA in liquid medium (Figure 7). These results confirmed the efficiency of marine-derived fungi in the biodegradation of persistent compounds and contributed to the improvement of decon‐ tamination techniques. Detailed results were published recently in the literature [134].

**Figure 7.** Proposed PCP biodegradation pathway by marine fungus *T. harzianum* DL2B (CBMAI 1677).
