Bioremoval capacity of phenol by some selected endophytic fungi isolated from Hibiscus sabdariffa and batch biodegradation of phenol in paper and pulp effluents

Abstract

Background and Objectives:

The use of endophytic fungi for management of phenol residue in paper and pulp industries has been shown as cost-effective and eco-friendly approach. In this study, isolation of endophytic fungi from roots, stems, and leaves of Hibiscus sabdariffa was conducted. Additionally, the isolated fungi were examined for their ability to degrade phenol and its derivatives in paper and pulp industrial samples, using different growth conditions.

Materials and Methods:

Out of 35 isolated endophyitc fungi, 31 were examined for their phenol biodegradation capacity using Czapek Dox broth medium containing Catechol and Resorcinol as a sole carbon source at final concentrations of 0.4, 0.6 and 0.8%.

Results:

A total of 35 fungal species belonging to 18 fungal genera were isolated and identified from different parts of H. sabdariffa plants. All strains have the capability for degrading phenol and their derivatives with variable extents. The optimum condition of degrading phenol in paper and pulp effluent samples by Fusarium poae11r7 were at pH 3–5, temperature at 28–35°C, good agitation speed at no agitation and 100 rpm.

Conclusion:

All endophytic fungal species can utilize phenol and its derivatives as a carbon source and be the potential to degrade phenol in industrial contaminants.

INTRODUCTION

Fungal endophytes represent a diverse group of unrelated filamentous fungi. They occur in most plants and often stay without causing any immediate, negative effects for the plant (1). Endophytes play a major role in the phytoremediation process and degradation of environmental toxins by direct or indirect process, indirectly by accelerate the phytoremediation process through promoting plant growth that has the ability of phytoremediation, or directly through degradation and/or accumulating pollutants by itself. Endophytes have the main role in the ecosystem are decomposers because they are the primary colonizers of dead plant tissues (2).

Hibiscus sabdariffa (Hs, roselle; Malvaceae) is an annual or perennial herb or woody subshrub and locally known as “karkade”, Hibiscus sabdariffa used as a diuretic, hypertensive, treating the viscosity of the blood and used traditionally as a portion of food, a herbal drink and as herbal medicine (3). In addition, previous study demonstrated that H. sabdariffa is potential source of endophytic fungi with good plant growth promoting properties and antimicrobial activity (4).

Phenol is an aromatic molecule and it is widely distributed naturally and present in the decay of organic materials or coal (5). Phenol is considered hazardous pollutants in many industrial methods such as petroleum strainers, pharmaceuticals, paper industries, textiles, plastics, dyes, and phenolic resin manufacturers (6). For example, the high antibacterial and inhibitory activities of phenols may generate problems in the operation of biological process. Additionally, phenols exhibited negative impacts on the quality of food processing and paper industry due to their odour and coloring properties (7).

Due to the cyclic molecular structure of phenol, the degradation rate is slow and difficult in the environment because of the difficulty of cleaving the benzene ring that causing their accumulation in the effluent environment (8). Even at low concentrations, phenolic compounds are highly toxic to living organisms and deadly to the aquatic system (9). Various treatments technique of wastewater containing high concentrations of phenol represents the most economical and environmental challenges to industries. Biotechnology is considered very effective in dealing with major environmental challenges through utilizing different chemical and physical methods but these methods are very expensive and may produce hazardous by-products. The biological process has been reported to be the most preferring and economical methods for the biodegradation of phenol (10). Biodegradation of phenol by the biological process is preferable to the physiochemical process because it is environmentally friendly and cost-effective (6). Several microorganisms can utilize phenol as carbon and energy sources for their growth (11). Most of the phenol-degrading microorganisms such as fungi including, Aspergillus fumigatus, Fusarium flocciferum, Aspergillus awamori, Penicillium chrysogenum, Trichosporium cutaneum, and Candida tropicalis have been proved as phenol degrader (12). Fungi can mineralize a wide variety of carbon sources to produce various active enzymes that providing possibilities for the removal of phenol and their derivatives (13). Many reports explained the mechanism of degradation of phenol and the metabolism of aromatic compounds and the mechanism of phenol biodegradation mainly takes place through Meta and Ortho pathways were illustrated (14). Several factors influence the rate of degradation of phenol including, temperature, pH, agitation, and physical properties of contaminants (15). The present study aimed to determine the biodiversity of endophytic fungi associated with Hibiscus sabdariffa and investigate the ability of some isolated endophytic fungal species to degrade different concentrations of phenol derivatives, Catechol (1,2 dihydroxy-benzene) and Resorcinol (1,3 dihydroxy benzene) and use of high biodegradation fungi in the degradation of phenolic compounds in the effluent from pulp and paper factory.

MATERIALS AND METHODS

Plant materials.

Healthy plant materials of Hibiscus sabdariffa were collected from a natural habitat in the desert of Aswan University Campus, Aswan governorate, Egypt which latitude: 24° 5’ 15” N32° and 53’ 56” E. Fifteen healthy medicinal plants were selected from five different sites in Aswan University area. The climatic environments in this region ranged between moderately to cold dry winter (16).

Isolation and identification of endophytic fungi from Hibiscus sabdariffa.

The collected samples from the plant material of Hibiscus sabdariffa (root, stem, and leaf) were completely rinsed twice in running tap water, then surface-sterilized by successive immersion in 70% ethanol (1 min) and then in 5% (v/v) sodium hypochlorite (5 min). Surface-sterilized roots stem, and leaves were longitudinally cut into approximately ∼5 mm segments under sterile conditions and directly placed on a sterilized Petri dishes containing potato dextrose agar supplemented with 100 U/mL chloramphenicol to suppress bacterial growth. Plates were incubated at 28°C for 2–3 wk until formed colonies. The growing tips of mycelia were transferred to new PDA plates for pure culture and maintained by continuous sub-culturing (17). All isolated fungal colonies were identified according to the microscopic observation of mycelia, asexual/sexual spores and colony morphology, cultural characteristics (color, texture, and pigmentation), and spores and spore-bearing structure using standard identification manuals such as Manual of Soil Fungi, The Genera of Hyphomycetes from Soil, Soil fungi in Qatar and other Arab Countries and Raper and Fennell for Aspergillus species (18–21). Colonization frequency (CF) was calculated as described by Hata et al. (22) using the following formula:

$$\text{CF}\%=\text{Number}\hspace{0.17em}\text{of}\hspace{0.17em}\text{segment}\hspace{0.17em}\text{colonized}\hspace{0.17em}\text{by}\hspace{0.17em}\text{endophytes}/\text{Total}\hspace{0.17em}\text{number}\hspace{0.17em}\text{of}\hspace{0.17em}\text{segments}\hspace{0.17em}\text{examined}\times 100$$

Biodegradation of phenolic compound by isolated endophytic fungi: Microorganisms.

Thirty-one strains of endophytic fungi that were isolated from H. sabdariffa were used for screening their ability for biodegradation of phenol and its derivatives at different concentrations.

Medium for degradation studies.

Czapek Dox broth medium was used during studies of the biodegradation of phenol by strains of endophytic fungi which had the following composition (g/L) sodium nitrate 3.0, potassium phosphate (dibasic) 1.0, potassium chloride 0.5, and magnesium sulfate heptahydrate 0.5. The pH of the medium was adjusted to 5.5 (23). Biodegradation tests were performed by supplemented the medium with Catechol (1,2dihydroxy-benzene) and Resorcinol (1,3 dihydroxy benzene) in concentrations of 4,6 and 8 (g/L) as sole carbon and energy sources.

Screening for phenol biodegradation potential.

Five ml of sterilized CzapekDox broth medium were inoculated with an 8 d culture in discs form (5 mm) from the plate as inoculums and the tubes were incubated under shake culture condition on a rotary shaker for 8 d at 28ºC. After an incubation period, the cells were centrifuged at 1200 rpm for 12 min. Residual phenol in the cell-free supernatants was estimated using Folin-Ciocalteu reagents method by taking about 100 μl of each supernatant and adding 100 μl of Folin-Ciocalteu reagents. The mixture was then allowed to stand for 5 min, then 100 μl sodium carbonate (2%) was added to the mixture and incubated for 60 min at 20ºC (24). The absorbance was measured at 725 nm. The residual phenol content was detected. The phenol removal efficiency of the tests strains was calculated according to the following formula:

$$\%\text{Phenol}\hspace{0.17em}\text{Removal}\hspace{0.17em}\text{Efficiency}\hspace{0.17em}(\text{PRE})=\left(\text{Ci}-\text{Cf}/\text{Ci}\right)\times 100$$

Where ci is the initial concentration of phenol (mg/L) and cf is the final concentration of phenol.

All experiments of phenol biodegradation were done triplicate; the results were expressed as average ± standard deviation (SD) (25).

Batch biodegradation of industrial effluent samples.

Six isolates of endophytic fungi that showed the highest degradative potential used for batch biodegradation of industrial effluent samples containing phenol, were selected, Aspergillus niger 13r7, Aspergillus japonicus 4r2, Alternaria chlamydospora 6l4, Cochliobolus australiensis 5l7, Emericella quadrilenata 1f7 and Fusarium poae 11r7.

Medium for batch biodegradation culture.

Industrial effluent samples were collected from the Paper and Pulp factory in the Edfu area in the Aswan government, Egypt. The industrial effluent samples were supplemented by adding necessary macro elements, KH₂PO₄ – 1.0 g/L, MgSO₄ – 0.50 g/L, KCL – 0.5 g/L, NaNO₃ – 3.00 g/L (26).

Screening of isolates with highest phenol batch biodegradation.

Each of six endophytic fungi were inoculated in 250 mL flasks containing 50 mL of industrial effluent medium samples and incubated at 28°C at 150 rpm on a rotary shaker for 5 d. Control assay (without inoculums) was performed under the same experimental conditions. Residual Phenol content was measured at various intervals over 5 d by using Folin-Ciocalteu reagents at 725 nm against a blank (24). All experiments of batch phenol biodegradation were done triplicate; the results were expressed as average ± standard deviation (SD). The total phenol contents were determined spectrophotometrically as described above using Folin-Ciocalteu reagent (24).

Factors influence on batch biodegradation rate of phenol.

Fusarium poae 11r7 were the most effective fungal species and have the highest and quickly batch biodegradation of phenol in industrial effluent samples, so it was selected for further optimal conditions experiments such as temperature, pH, different agitation speed, and incubation time during 5 d to detect the best optimization conditions for highly degradation rate. Experiments were performed in 250 mL Erlenmeyer flask containing 100 mL of Czapek Dox broth medium and autoclaved at 121°C for 15 min then supplemented with industrial effluent samples after medium sterilization. Disc of Fusarium poae 11r7 of 5 d old culture on Czapek Dox agar medium was used as inoculums on the tested medium. The above growth medium supplemented with industrial effluent samples without F. poae 11r7 inoculum has been used as control. Conditions parameters were varied: for temperature (28, 35, and 45°C), for pH (3.0, 5.0, 7.0, and 9.0 using 1.0 M HCl or 1.0 M NaOH, for incubation at without agitation and agitation at different speeds (100, 150, and 200 rpm) on different 3 shakers and for different incubation time during 5 d (1, 2, 3, 4, and 5 d) (25). Folin-ciocalteu reagent method was used for the estimation of residual phenol content (24). All experiments of phenol biodegradation were done triplicate; the results were expresses as average ± standard deviation (SD).

Statistical analysis.

Data obtained were subjected to a one-way analysis of variance (ANOVA). Means were compared using Tukey’s test analysis at the 5% level using Minitab software. The values shown in the figures were carried out by using the R program (R-3.4.3, https://www.r-project.org/).

RESULTS

Endophytic fungi associated with H. sabdariffa.

From 15 individual plants corresponding to Hibiscus sabdariffa (roots, stems, and leaves) plants were collected from different places for the study of endophytic fungi. A total of 34 fungal species belonging to 18 fungal genera in addition to one variety were isolated and identified from Hibiscus sabdariffa plants. The results showed that the root tissue of Hibiscus sabdariffa harbors the largest number of endophytic fungi (26 fungal isolates) as compared to the stem (10 fungal isolates), whereas only 16 fungal isolated from the leaves. The Aspergillus genus was the most dominant fungal genus in all plant parts of H. sabdariffa (38.5%) followed by the Chaetomium genus (10.88%) and Macrophomina phaseolina (Fig. 1B) In this study, the relative abundance of the Aspergillus genus in the different parts of the plant was 22.6%, 10.46%, and 5.44% of the roots, stem, and leaves, respectively, and represented by seven species. The distribution of endophytic fungal species in different parts of the plant was variable, whereas Aspergillus jabonicus and Macrophomina phaseolina were the most common fungal species in the root (24 colonies and 23 colonies respectively). Aspergillus niger and Cochliobolus australiensis in the stem (14 colonies and 8 colonies respectively). Cladosporium sphaerospermum and chaetomium elatum were common in the leaf (10 colonies and 7 colonies respectively) (Fig 1A, 1C, 1D).

Fig. 1.

Diversity profile of endophytic fungi associated with H. sabdariffa, A Total count of fungal species, B% Genera Dominance of fungal species. C Venn diagram of the total number and mutual occurrence of fungal species of the root, stem, and leaf, D heat map illustrate % Colonization frequency (CF) of fungal species

Biodegradation of phenol by endophytic fungi from H. sabdariffa.

In this present study, 31 endophytic fungal species representing 91.18% of the total 34 isolates have the capability of biodegradation of two phenol derivatives, Catechol (1,2 dihydroxy-benzene and Resorcinol (1,3 dihydroxy benzene) at three concentrations (0.4, 0.6 and 0.8%, w:v) with variable extents.

As shown in Table 1, the highest degradation of the fungal strains in range between (75.85 to 97.85%, 57.63 to 98.50%, and 10.34 to 83.45%) at 0.4%, 0.6% and 0.8% concentration of 1,2 dihydroxy benzene respectively. The highest degradation rate by 97.85 and 97.82% of 1,2 dihydroxy-benzene at final concentration of 0.4% was observed in Emericella quadrilenata and Cochliobolus australiensis 9r2, respectively (Table 1). On the other hand, Emericella nidulans var. echinulata showed the lowest degradation rate by 75.63% of 1,2 dihydroxy benzene (Table 1). With increasing of concentration of 1,2 dihydroxy-benzene (catechol), the capability of endophytic fungal species differed with increasing or decreasing in the ability of biodegradation. There were decreasing in the rate of biodegradation in all species such as Cochliobolus australiensis 9r2 (95.30%), Macrophomina phaseolina (95.50%) except in Aspergillus flavus 13r3, Aspergillus japonicus 1r2, Aspergillus oryzae 33r5, Emericella quadrilenata 1f7, Emericella nidulans var. echinulata, Emericella sp. 3r3, Fusariun solani 22r5 and Phoma leveilei which had increasing in the biodegradation but with increasing of catechol concentration to 0.8%, there were decreasing in biodegradation rate in all fungal strains.

Table 1.

Potency of different fungal species on percentage degradation of Catechol at 0.4, 0.6, 0.8 % concentrations, incubated at 28ºC for 8 d

Species of endophytic fungi	0.4	0.6	0.8
Alternaria chlamydospora 6l4	97.01 ± 0.08AB	94.58 ± 0.89ABC	59.68 ± 1.03EFGHI
Aspergillus flavus 12r5	85.95 ± 0.76GHI	74.46 ± 0.73L	49.68 ± 1.20IJKL
Aspergillus flavus 13r3	92.41 ± 0.37D	92.63 ± 0.72BCD	55.08 ± 2.16GHIJK
Aspergillus fumigates 27l2	84.23 ± 0.17IJ	73.80 ± 0.32L	44.48 ± 2.28KLM
Aspergillus japonicus 1r2	86.10 ± 1.13GHI	90.17 ± 0.50CDE	10.34 ± 1.99S
Aspergillus japonicus 4r2	94.06 ± 0.93CD	92.24 ± 0.09BCD	23.97 ± 0.23P
Aspergillus niger 13r7	95.38 ± 0.07BC	92.48 ± 0.23BCD	28.48 ± 5.75OP
Aspergillus oryzae 33r5	93.76 ± 0.08CD	93.77 ± 0.50BC	79.35 ± 2.28ABC
Aspergillus oryzae 7r3	81.84 ± 0.51KL	81.35 ± 2.23IJK	20.67 ± 4.27QRS
Aspergillus terreus 1r7	81.84 ± 0.51KL	66.02 ± 3.22M	76.38 ± 0.07ABC
Aspergillus terreus 26l2	79.14 ± 0.83M	77.60 ± 1.31KL	59.26 ± 0.68FGHI
Asperhillus terreus 1s6	92.02 ± 0.81DE	88.86 ± 0.63DEF	45.59 ± 2.62JKLM
Chaetomium elatum 6s6	89.72 ± 0.54EF	85.32 ± 0.96FGHI	61.36 ± 1.25EFGH
Chaetomium homopilatum 33l5	92.45 ± 0.07D	82.26 ± 0.51	70.05 ± 1.54CDEF
Cochliobolus australiensis 5l7	86.57 ± 0.88GH	79.70 ± 0.24JK	83.45 ± 0.97A
Cochliobolus australiensis 8r2	93.70 ± 1.37CD	88.43 ± 0.03DEFG	75.35 ± 1.77ABCD
Cochliobolus australiensis 9r2	97.82 ± 0.64A	95.30 ± 0.36AB	37.22 ± 2.28MNO
Drechslera bioseptata 18r2	95.11 ± 0.29BC	92.57 ± 0.40BCD	70.89 ± 0.51BCDE
Emericella nidulans var. echinulata 30s5	75.85 ± 0.03LM	86.07 ± 0.10EFGH	81.45 ± 2.62AB
Emericella nidulans var. echinulata 31s5	80.40 ± 0.54N	84.39 ± 0.46FGHI	69.91 ± 0.86CDEF
Emericella quadrilenata 1f7	97.85 ± 0.03A	98.50 ± 0.64	40.89 ± 3.25LM
Emericella ruglosa 3s4	89.45 ± 0.30F	88.72 ± 0.86DEF	34.15 ± 0.22NOP
Emericella sp. 3r3	79.23 ± 0.24M	85.79 ± 0.55EFGHI	55.40 ± 2.00GHIJK
Fusarium poae 11r7	85.54 ± 0.64GHIJ	83.99 ± 0.17GHIJ	64.89 ± 0.40DEFG
Fusarium solani 22r5	83.38 ± 0.05JK	85.58 ± 0.53FGHI	46.52 ± 3.06JKLM
Helicosporium sp.	86.03 ± 0.90GHI	76.97 ± 0.84KL	38.48 ± 0.57LM
Humicola hyalothermophila	97.12 ± 0.45AB	94.17 ± 0.28ABC	52.99 ± 4.78HIJK
Macrophomina phaseolina	96.52 ± 0.35AB	95.50 ± 0.74AB	82.05 ± 0.07AB
Penicillium expansum	89.58 ± 0.26F	57.63 ± 3.95N	45.03 ± 10.16JKLMN
Phoma leveilei	84.39 ± 0.43HIJ	96.58 ± 0.13AB	14.34 ± 0.91RS
Thanatephorus cucumeris	86.70 ± 0.33G	86.60 ± 0.13EFGH	55.96 ± 1.42GHIJ

As shown in Table 2, the highest degradation of the fungal strains occurred when they used Resorcinol (1,3 dihydroxy benzene) as a carbon source in the range between (94.54 to 99.85%, 97.93 to 99.67% and 83.30 to 99.04%) at 0.4% 0.6%, and 0.8% concentrations. The highest degradation rate by 99.85% and 99.66% of 1,3 dihydroxy-benzene (Resorcinol) at final concentration of 0.4% was observed in Aspergillus fumigatus 27l2 and Aspergillus flavus 12r5, respectively (Table 2). On the other hand, Alternaria chlmydosora 6l4 showed the lowest degradation rate by 94.54. With increasing the concentration of 1,3 dihydroxy-benzene (Resorcinol) to 0.6%, the ability of endophytic fungal species differed with increasing or decreasing in the ability of biodegradation. There were decreasing in the rate of biodegradation in all species except in Alternaria chlmydosora 6l4, Aspergillus terreus 1r7, Cochliobolus australiensis 8r2, Cochliobolus australiensis 9r2 and Macrophomina phaseolina which had increasing in the biodegradation. The tested endophytic fungal species can degrade phenol up to a concentration of 0.8%. The limit of phenols in paper and pulp industries was 22 mg L⁻¹.

Table 2.

Potency of different fungal species on percentage degradation of Resorcinol at 0.4, 0.6, 0.8% concentrations, incubated at 28ºC for 8 d

Species of endophytic fungi	0.4	0.6	0.8
Alternaria chlamydospora 6l4	94.54 ± 0.70JK	97.06 ± 0.06BCDE	90.32 ± 1.68GHIJ
Aspergillus flavus 12r5	99.66 ± 0.14AB	92.31 ± 0.97KLMN	90.77 ± 0.08FGHI
Aspergillus flavus 13r3	95.37 ± 0.63IJK	93.82 ± 0.37HHIGKLM	92.92 ± 0.59DEF
Aspergillus fumigates 27l2	99.85 ± 0.00A	94.73 ± 0.68EFGHIJ	91.07 ± 0.10FGHI
Aspergillus japonicus 1r2	96.39 ± 0.30GHI	89.81 ± 1.00OP	89.68 ± 0.45HIJ
Aspergillus japonicus 4r2	97.32 ± 0.61EFGH	79.93 ± 0.41R	85.55 ± 0.20KL
Aspergillus niger 13r7	97.52 ± 0.90CDEFGH	97.41 ± 0.30ABCD	80.55 ± 1.27M
Aspergillus oryzae 33r5	98.45 ± 0.69ABCDEF	91.56 ± 0.63MNO	96.38 ± 0.14B
Aspergillus oryzae 7r3	98.72 ± 0.22ABCDEF	94.19 ± 0.80GHIJKL	84.25 ± 0.06L
Aspergillus terreus 1r7	94.35 ± 0.73K	95.20 ± 0.14DEFGHI	94.00 ± 1.35BCDE
Aspergillus terreus 26l2	98.49 ± 0.59ABCDEF	92.12 ± 0.94LMNO	89.16 ± 0.60IJ
Asperhillus terreus 1s6	99.22 ± 0.17ABCD	97.46 ± 0.80ABCD	84.12 ± 0.66L
Chaetomium elatum 6s6	99.02 ± 0.19ABCDE	96.78 ± 0.11BCDEF	95.61 ± 0.14BC
Chaetomium homopilatum 33l5	98.83 ± 0.40ABCDEF	96.60 ± 1.00BCDEF	92.31 ± 0.14EFG
Cochliobolus australiensis 8r2	98.73 ± 0.40ABCDEF	98.97 ± 0.91AB	95.07 ± 0.24BCD
Cochliobolus australiensis 5l7	97.87 ± 0.03BCDEFG	87.50 ± 0.43P	99.20 ± 0.14A
Cochliobolus australiensis 9r2	97.87 ± 0.03BCDEFG	98.32 ± 0.40ABC	91.55 ± 0.45EFGHI
Drechslera bioseptata 18r2	97.89 ± 0.04BCDEFG	96.32 ± 0.12CDEFG	99.04 ± 0.48A
Emericella nidulans var. echinulata 31s5	98.24 ± 0.88ABCDEF	95.69 ± 0.53DEFGH	95.02 ± 1.21BCD
Emericella nidulans var. echinulata 30s5	97.03 ± 0.03FGHI	94.57 ± 0.37FGHIJK	93.64 ± 0.90CDE
Emericella quadrilenata 1f7	95.79 ± 0.62HIJK	89.74 ± 0.57OP	83.72 ± 0.49L
Emericella ruglosa 3s4	97.36 ± 0.30EFGH	94.13 ± 0.86GHIJKL	87.82 ± 0.14JK
Emericella sp. 3r3	99.50 ± 0.07AB	98.48 ± 0.14ABC	94.02 ± 0.27BCDE
Fusarium poae 11r7	98.95 ± 0.25ABCDEF	92.87 ± 0.34IJKLM	98.92 ± 0.49A
Fusarium solani 22r5	98.56 ± 0.52ABCDEF	89.93 ± 0.97NO	85.33 ± 0.02KL
Helicosporium sp.	99.31 ± 0.08ABC	96.71 ± 0.74BCDEF	91.12 ± 0.22FGHI
Humicola hyalothermophila	99.25 ± 0.03ABC	96.22 ± 0.06CDEFG	85.43 ± 0.41KL
Macrophomina phaseolina	99.12 ± 0.30ABCDE	99.67 ± 0.09A	98.95 ± 0.12A
Penicillium expansum	99.00 ± 0.72ABCDE	98.18 ± 0.63ABC	92.15 ± 0.16EFGH
Phoma leveilei	96.32 ± 0.14GHIJ	86.64 ± 0.23Q	83.30 ± 0.20L
Thanatephorus cucumeris	97.43 ± 0.55DEFGH	92.75 ± 0.37JKLM	93.65 ± 1.13CDE

All values are mean ± SD, Values in the same column with different superscript letters are significantly different, P < 0.05 (ANOVA after Tukey’s test analysis).

Batch biodegradation of industrial effluent sample by endophytic fungi.

A batch cultivation experiment was carried out using phenol as limiting substrate for 6 endophytic fungi named Alternaria chlmydosora 6l4, Aspergillus japonicus 4r2, Aspergillus niger 13r7, Cochliobolus australiensis 5l7, Fusarium poae 11r7 and Emericella quadrilenata 1f7. Initial phenol concentration in industrial effluent medium was taken as 278 mg/L. The extent of phenol concentration was investigated for several batch residence times by intermittent sampling, showing the biodegradation potential of six selected endophytic fungi in degrading synthetic phenol waste. There were decrease in the concentration of phenol during 5 days, in 2^nd day, Fusarium poae 11r7 considered the most effective fungi in decreasing the phenol percentage (by 37.4%) followed by Aspergillus japonicus 4r2 (by 42.34%), while the lowest phenol percentage (71.82%) was observed in Cochliobolus australiensis (Fig. 2). In 3^rd, 4^th, 5^th the value of phenol concentration decrease gradually until reach to 5^th day that there were no any phenol content in all six species. Reduction in phenol level proved the biodegradation. The highest amounts of phenol degradation were observed on 5 day while Emericella quadrilenata 1f7 consume all phenol content in the fourth day. Results from this study showed that Fusarium poae 11r7 has potential to be used in biodegradation of industrial effluent containing phenol.

Fig. 2.

Batch biodegradation of phenol in Paper and Pulp Effluent samples by some species of endophytic fungi during 5 d.

Factors influence on batch biodegradation rate of phenol.

Numerous factors are affecting the degradation rate of phenolic compounds such as temperature, pH, agitation speed, and incubation period. Fusarium poae 11r7 has been selected due to its high batch biodegradation rate of industrial effluent samples containing phenol. As shown in Fig. 3, temperature has significant impact on degradation efficiency of the selected fungal strains, Fusarium poae 11r7 with optimal temperature in the range of 28–35°C. The most suitable pH value for the selected fungal strain Fusarium poae 11r7 in increasing the batch biodegradation rate was pH 5, pH 3, and pH7 respectively for 5 d while pH 9 showed the lowest batch degradation rate.

Fig. 3.

Effect of different factors on the degradation rate of phenol in paper and pulp effluent samples by using Fusarium poae

The incubation of batch biodegradation of Fusarium poae 11r7 at static condition and agitation speed of 100 rpm showed increasing in biodegradation rate of phenol due to decreasing the absorbance of phenol. With increasing the agitation speed to 150 and 200 rpm, there were decreasing in batch biodegradation rate observed. The different incubation periods on the degradation rate of phenol by Fusarium poae 11r7 resulted the increment of degradation rate of phenol with increasing the incubation period (decreasing in absorbance). At the 5 d, maximum phenol degradation occurred because of the maximum utilization of all content of phenol.

DISCUSSION

The relationship between endophytes and host plants involves both antagonism and mutualism. Plants have many mechanisms to limit the growth of endophytes which include producing a variety of toxic metabolites. But by the time of co-evolution, endophytes have gradually formed a variety of tolerant mechanisms towards host metabolites by producing exoenzymes and mycotoxins. These enzymes may also degrade macromolecule compounds into small molecules or convert more toxic substances into less toxic to increase their adaptability (27). A total of 35 fungal species belonging to 18 fungal genera were isolated and identified from different parts of Hibiscus sabdariffa plants. Aspergillus was the most dominant genus followed by Chaetomium genus and Macrophomina phaseolina. Aspergillus genus was isolated with 7 species that appeared in all parts of plants followed by 3 species of Chaetomium genus and Machrophomina phaseolina that is in agreement with the study of Kamel et al. (28) who isolated five species of Aspergillus and two varieties of A. terreus and 2 species of Chaetomium genus and Machrophomina phaseolina as endophytic fungi from Euphorbia geniculata at the same region of our study. Likewise, Aspergillus genus exhibited the highest colonization frequency among different endophytic fungi isolated from H. sabdariffa (4). Aspergillus species are characterized by high secretion of secondary metabolites with various biological activities which may attributed to high dominance of Aspergillus genus in H. sabdariffa (29).

Nath and Joshi (4) also isolated eight endophytic fungi from different parts of the plant H. sabdariffa L., mostly being isolated from the leaf segments. Patil et al. (30) isolated different endophytic fungal species from variable medicinal plants such as Alternaria spp., Rhizopus spp., Curvularia spp., and Trichoderma spp. Biodiversity of endophytic fungal species was reported such as Aspergillus sp., Alternaria sp., Penicillium sp. and Fusarium sp. that were isolated from Camellia oleifera (31). Aspergillus sp., Alternaria sp., Chaetomium sp. and Cladosporium sp. were isolated from traditional medicinal plants (32).

Phenol and its derivatives are principle pollutants that are highly toxic even at low concentrations in the industrial effluent and are considered one of the largest groups of environmental pollutants due to their presence in many industrial effluents (8). In the past few years, there has been several reports that focused on the capability of fungi in degrading phenol and its derivatives and used as a novel for degradation of environmental pollutants including hydrocarbons, polychlorinated biphenyl’s (PCBs), polyaromatic hydrocarbons (PAHs), and metals (27). Biodegradation of phenol by fungi is influenced by several factors such as the substrate concentration that considered as an important factor in phenol biodegradation. Also phenol itself is well known to inhibit microbial growth, especially at greater concentrations. This phenomenon is commonly known as substrate inhibition. The high concentration of phenol can affect the growth of microorganisms, and the concentration at which inhibition occurs varies from one compound to another (10). All endophytic fungal strains have the capability for degrading phenol and their derivatives at different concentrations (0.4%, 0.6% and, 0.8%) with variable extents. Stoilova et al. (33) studied biodegradation of phenol and other derivatives by Aspergillus awamori at different concentration (0.3, 0.6. 1,2 and, 3 g/L) and revealed that the fungal strain utilized phenol with the varying extent depending on phenol concentration and the rate of degradation decreased gradually with increasing the phenol concentration above 0.6 g/L. Santos et al. (34) and Kennes and Lema, (35) reported the fungal strains, Graphium sp. and Phanerochaete chrysosporium were significantly degrade phenol at concentration of 0.3 and, 0.05 g/L, respectively. In the present study, the endophytic fungal species could degrade phenol up to a concentration of 0.8%. Stoilova et al. (12) studied the rate of degradation of catechol, 2,4-dichlorophenol and 2,6-dimethoxyphenol with different concentrations (0.1%, 0.2% and 3.0%) by Aspergillus awamori. The capability of tested endophytic fungal species on the degradation of phenol and its derivatives decreased or increased with increased the concentration of phenol. The capability of different species of endophytic fungi on the degradation of phenol and its derivatives differed from species to species and from derivatives to derivatives.

In batch biodegradation studies, Fusarium poae 11r7 was able to degrade phenol in paper and pulp effluent samples with an initial concentration of 278 mg/L to 104 mg/L within 5 d of incubation period. Sharma and Gupta (36) revealed that Aspergillus niger was able to degrade phenol content of paper and pulp effluent samples with an initial concentration of 268 mg/L to 119 mg/L after five d of incubation. Bernats and Juhna (8) and Lu et al. (37) studied batch biodegradation of phenol in pharmaceutical and coking wastewaters by fungi, respectively. Chhaya and Gupte (38) studied the capability of degrading phenolic compounds olive mill wastewaters by 89% in 5 d.

In batch biodegradation of Fusarium poae 11r7, the optimal temperature with highest biodegradation rate was 28–35°C. The phenol biodegradation rate decreased rapidly at temperature up to 35°C because the catalytic activity of the enzymes started to decrease over that temperature. The optimal pH for phenol biodegradation by Fusarium poae 11r7 was found to be at pH 3–5 and 7. Sharma and Gupta (36) studied the influence of different pH values on the biodegradation rate of phenol and indicated that the maximum phenol degradation occurs at neutral pH. At acidic or basic pH there was decreasing in phenol degradation since the culture utilizes less carbon source. Bernats and Juhna (8) studied factors affecting the biodegradation of phenol and indicated that the optimal conditions (pH and temperature) of biodegradation were at pH 5–6 and temperature 25°C. Briefly, the optimum conditions for phenol degradation with Fusarium poae 11r7 were found at pH 3–5 and temperature 28–35°C. These results indicated that the pH and temperature parameters are important factors for fungi growth and subsequently, increasing the efficacy of phenol biodegrading.

Static condition and agitation speed of 100 rpm showed increasing in the degradation rate of phenol by Fusarium poae 11r7 due to decreasing the absorbance of phenol. Maximum phenol degradation has occurred on the 5 d because of the maximum utilization of all content of phenol. Santos and Linardi (5) reported that Graphium, Aspergillus, Fusarium and Penicillium can utilize aromatic and aliphatic hydrocarbons and Graphium sp. FLB4 degraded 75% of 10 mM of phenol in 168 h.

CONCLUSION

In this study, 34 endophytic fungi were isolated from different parts of the medical plant H. sabdariffa. All the isolated endophytic fungi were screened for their ability to degrade phenol residues in the industrial pulp samples. Out of them, 31 fungi species showed considerable ability to degrade phenol derivatives. The potency of biodegradation of phenol derivatives differed from species to species and from phenol derivatives to another. In general, six endophytic fungal species were found to have significant activity in biodegrading industrial effluent samples containing phenol. Fusarium poae 11r7 considered the most effective fungi in decreasing the phenol percentage by 37.4% in batch biodegradation, thus F. poae 11r7 considered as the potential candidate for the biodegradation of industrial effluent samples containing phenol. The optimum temperature and pH for Fusarium poae 11r7 were found to be 28–35°C, and 3–5, respectively. Static condition and agitation rate 100 rpm were found to be the optimal conditions for biodegradation of phenol.