A novel approach of using Ash gourd as a substrate for enhancing lingo-cellulolytic enzyme production in mono and co-culture of two white rot fungi

Introduction

It is already known that basidiomycetes fungal isolates with higher secretion of ligninolytic enzymes as well as enzymes having unique capacity to be used for their biotechnological, environmental and industrial application (Kiiskinen et al., 2004; Mikiashvili et al., 2004) [19,27] . But implementing of these potential applications of enzymes at industrial scale needs large amount of these enzymes which should have been coast effective also. So, selection of the organisms having ability to produce such enzymes in greater amount needs to be carried out. White-rot fungi (basidiomycetes) are having distinctive ability to degrade all components of wood due to their competency to produce cell wall degrading enzymes. Almost all the enzymes produced are extracellular. Which are oxidative and hydrolytic in nature. So, they are also known as lignocellulolytic enzymes which are responsible for degradation of lignocellulose (Liu et al., 2013a, van den Brink and de Vries, 2011, Bissaro et al., 2018) [2,34,5] . The fungi produce one or more of three extracellular enzymes that are important for lignin degradation: lignin peroxidase (EC 1.11.1.14), manganese-dependent peroxidase (EC1.11.1.13), and laccase (EC 1.10.3.2). Some white rot fungi secrets all these enzymes while others produce only few of them. The major hydrolytic enzymes are endo-1, 4-β-Dglucanase (EC 3.2.1.4), exo-1, 4-β -D-glucanase (EC 3.2.1.91), and xylanase (EC 3.2.1.8). Several methodologies like use of mixed culture (co-culture) of organisms, genetically modified organisms, use of some lignocellulosic waste material or chemical enhancers can results in higher production of such enzymes. Lignocellulosic material is rich in soluble carbohydrates and chemical inducers. So, intermediate compounds of enzyme synthesis helps in enhancing production of these enzymes (Elisashvili et [10,31] . The selection of lignocellulosic materials and its concentration played an important role in promoting fungal growth and targeted enzyme synthesis (Kapich et al 2004 and Rosales et al 2007) [18,32] . Several raw materials have been used successfully for enhancing lignocellulolytic enzyme production in submerged (SF) and solid-state (SSF) fermentation (AboSiada et al 2017, Kachlishvili et al. 2006; Elisashvili et al. 2006, 2008; Rosales et al. 2007; Osma et al. 2007; Levin et al. 2007) [1,15,10,32,30,21] . Ash gourd (Benincasa hispida Cogn.) is cultivated as a vegetable throughout India, in other Southeast Asian countries, and Japan, China, and Australia. Ash gourd is known to have a high concentration of carbohydrates, especially xylan (Mazumdar et al., 2004) [25] and pectin (Mazumdar et al., 2005) [26] . The present study evaluates the effect of a co-culture of two compatible white rot fungi and various concentrations of ash gourd pulp on the lignocellulolytic enzyme production using submerged fermentation process.

Material and Methods

Fungal cultures and maintenance

Fungal isolates of Irpex lacteus and Daedaleopsis confragosa were procured from Forest Research Institute, Dehradun. On the basis of paired interaction tests performed in the laboratory Irpex lacteus and Daedaleopsis confragosa were compatible with each other and so selected for co culture experiment. Cultures were maintained on Malt Extract Agar (MEA) medium at 4 (±1) 0 C in Seed Anatomy laboratory, Department of Botany, The Maharaja Sayajirao University of Baroda, Gujarat, India.

Lignocellulosic substrate preparation

Ash gourd was acquired from the local market of Vadodara, Gujarat. The peel and seeds of Ash gourd were removed. Only the pulp portion was cut in to small pieces and oven dried at 80˚ C.

Preparation of fungal enzyme sample

Enzyme assays were executed in 100 ml of 3% Malt extract broth (MEB) medium. The MEB medium was sterilized by autoclaving. After cooling, inoculation was inoculation was performed with 9 mm disc of 10 days old culture in aseptic condition and for co-culture/dual culture 9 mm agar disc of both the selected fungal culture were inoculated in the Erlenmeyer flask of MEB medium. To estimate the effect of various concentration of ash gourd the dried pieces of ash gourd were augmented in the 100 ml of above mentioned medium (1%, 2%, 3%, 4%, 5%, 6%, 8% and 10%). The culture was incubated at 25 ±1˚ C for the desired incubation period. After completion of incubation period the cultures were removed, mycelium was homogenized in a Waring laboratory blender and filtered through whatman paper No. 1 disc. This crude filtrate was used to evaluate the activity of enzymes.

Enzyme assay Lignin peroxidase (LiP) and Manganese-dependent peroxidase (MnP)

Lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities were estimated according to the procedure of (Castillo et al., 1997) [7] using 0.167 mM 3methyl-2-benzothiazolinone hydrazone (MBTH) as substrate which interact with 2.37 mM 3-(dimethylamino) benzoic acid (DMAB) producing a purple coloured reaction. Substrates were prepared on 0.1 M succinic-lactic acid buffer at pH 4.5. Reaction mixture contained 417.5 µl MBTH, 417.5 µl DMAB, 100 µl MnSO 4 , 50 µl supernatant (enzyme) and 15 µl H 2 O 2 to measure the enzyme activity. To discriminate between magnesium dependent and independent peroxidases, reaction was performed in the presence and absence of 3mM MnSO 4 . 4mM H 2 O 2 respectively to initialise the reaction. Reactions were carried out at 37°C and monitored spectrophotometrically at 590 nm. The enzyme activity was calculated using extinction coefficient ɛ = 53000 M -1 cm -1 .

Laccase

Laccase activity was examined using 2, 2'-azino-bis (3ethylbenziazoline-6-sulphonic acid) (ABTS) as a substrate (Niku-paavola et al., 1988) [29] . Reactions were carried out with 500 µM ABTS in 50 mM Sodium acetate buffer at pH 4.5. A 20 µl aliquot of enzyme solution was added to 580 µl of the ABTS. Absorbance at 420 nm were observed and the enzyme activity was calculated using extinction coefficient (ɛ = 36, 000 M -1 cm -1 ).

Aryl alcohol oxidase (AAO)

Aryl alcohol oxidase (AAO) activity was estimated using Veratryl alcohol as substrate (Guillen et al., 1990) [13] . The reaction mixture contained 5mM veratryl alcohol in a 0.1 mM sodium phosphate buffer pH 6 and 50 µl enzyme solution. Reaction was performed at 30°C and visualized spectrophotometrically at 310 nm and the enzyme activity was calculated using extinction coefficient (ɛ = 9300 M -1 cm - 1 ).

Xylanase

The amount of xylanase produced was measured by using 1% birch wood xylan as the substrate (Bailey et al., 1992) [3] . Xylanase activity was assayed in 3.0 ml of a reaction mixture containing 0.1 ml of crude extracellular enzyme sample, 0.5 ml of 1% birch wood xylan (prepared in 0.05 M Na-citrate buffer, pH 5.3). The mixture was incubated at 55°C for 30 min. The reaction was stopped by the addition of 0.6 ml of 3, 5-dinitrosalicylic acid (DNSA) (Miller, 1959) [28] and 1.8 ml of distilled water was added to the tubes to make the reaction mixture 3 ml. The contents were boiled for 15 min (Miller, 1959) [28] . After cooling, the color developed was read at 540 nm. The amount of reducing sugars liberated was quantified using xylose as standard. One unit of enzyme activity is defined as the amount of enzyme which releases 1 µmol of xylose in 1 min under assay conditions (Khan et al., 1986) [20] .

Cellulase

Cellulase (CMCase) activity was determined by mixing 0.5 ml of 1% (w/v) CMC (prepared in 0.05 M acetate buffer pH 5.3) with 0.1 ml of crude extracellular enzyme solution incubating at 50°C for 30 min (Casimir et al., 1996) [6] . The reaction was stopped by the addition of 0.6 ml of 3, 5dinitrosalicylic acid (DNSA) (Miller, 1959) [28] and 1.8 ml of distilled water was added to the tubes to make the reaction mixture 3 ml. The contents were boiled for 15 min. The colour developed was read at 540 nm. The amount of reducing sugar liberated was quantified using glucose as standard. One unit of cellulase is defined as the amount of enzyme that liberates 1 µmol of glucose equivalents per minute under the assay conditions (Mandels et al., 1981) [24] .

Results and Discussion

In the present study assessment and comparison of the ligninolytic enzymes Lignin Peroxidase (LiP), Manganese Peroxidase (MnP), Laccase, Aryl alcohol oxidase (AAO), Xylanase and Cellulase in monocultures and co cultures of I. lacteus and D. confragosa at diverse incubation period were carried out and the results obtained are represented in Table 1. The optimum days for maximum production of the enzyme was also observed. Further effect of various concentration of ash gourd pulp was also evaluated on the day when highest enzyme activity was obtained. There was a variation in the enzyme activity of mono culture and co culture. Graphical representation of the enzymatic activity is represented in Fig 1 . The gradual increase in the enzyme activity was observed with increase in incubation time and once the maximum enzyme activity was achieved a gradual decries in the enzyme activity was observed. The greatest enzyme activity of all the enzymes except xylanase was observed on 25 th day and 30 th day in mono cultures of D. confragosa and I. lacteus respectively. The maximum xylanase activity was noticed on 20 th day after inoculation in both the monoculture. It was also observed that in co-culture of D. confragosa and I. lacteus the enzymes produced were more in amount, compared to its monoculture and also at less incubation days. The maximum secretion of all the lignin cellulolytic enzymes were noted on 20 th day whereas xylanase produced highest amount on 15 th day. Cellulase enzyme activity was observed to be absent throughout the experimental set up and I. lacteus did not showed LiP activity. Average enzyme activity determined from three replicates after respective incubation period in ±S.D.

In mono culture the highest LiP activity was obtained by DC 2.47U/L whereas IL showed lack of Lip activity and their coculture showed 2.74U/L LiP activity which was slightly elevated compared to monocultures (Fig. 1 A). MnP activity was observed in the range of 0.15 U/L to 1.97 U/L. In mono culture the optimum MnP activity was produced by DC (1.75U/L) followed by IL (0.69U/L) while coculture produced 1.97 U/L (Fig. 1 B). This result designates that in coculture more than two fold increase was noted in comparison with IL. Highest Laccase activity was secreted by IL (1.94 U/L) followed by DC 1.07U/L and coculture showed 5.45 U/L (Fig. 1 C) representing fivefold increase in laccase enzyme activity in coculture. Highest AAO activity between the monoculture was found in IL (36.95 U/L) followed by DC (24.42U/L) and coculture exhibited higher amount of enzyme activity (49.12 U/L) (Fig. 1 D). The maximum xylanase enzyme activity was observed in IL (16.87U/ml) followed by DC (13.86 U/ml) and co-culture produced (22.68U/L) (Fig. 1 E). Cellulase enzyme activity was found to be absent in monoculture and coculture (Fig. 1 F).

In the experimental studies of Hong et al 2012 on Lip activity it was observed that four different white rot fungi (Pleurotus ostreatus, Coriolus versicolor , Tyromyces albidus and Trametes gallica) in static submerged fermentation using peat as a substrate indicating some white rot fungi may not be producing LiP activities. Kamitsuji et al., 2004 [17] also demonstrated that Pleurotus species have been documented to produce no typical LiP activity. Absence of LiP activity was detected in the C. versicolor cultured medium using Olive mill wastewater as the substrate (Ergul et al., 2009) [12] . For ligninolytic enzymes, some white rot fungi produce all of these enzymes while others produce only one or two of them. Present study also demonstrated that IL showed absence of LiP activity. Maximum MnP and Laccase activity was detected in the co culture of IL+DC where in Laccase activity 5.45 U/L while in mono culture IL and DC produced 1.94 and 1.07 U/L respectively. MnP enzyme activity observed in co cultures of IL+DC was 1.97 and in mono cultures of IL and DC showed 0.75 and 1.75 U/L respectively which is in agreement with the observations of Chi et al. (2007) [8] who has reported that enzyme laccase and MnP secretion was enhanced in co-cultures of Pleurotus ostreatus and Ceriporiopsis subvermispora / Physisporinus rivulosus. Baldrian, 2004 also observed enhancement of Laccase enzyme in the co culture of Trametes versicolor and Pleurotus ostreatus. In Malt extract medium maximum lingocellulolytic enzymes like laccase and MnP production is attributed as malt extract provides the complete pool of amino acids required for enzyme synthesis (Arora & Sandhu 1985) [2] . Ma et al 2011 described effect of co culture treatment of Irpex lacteus and Auricularia polytricha on lignocellulosic degradation which showed highest activity on the 20 th day which was higher than observed in the mono cultures of both fungi which was exactly similar as the present study. In the present study both the fungi in monoculture and coculture showed variable amount of xylanase production and an absence of cellulase enzyme secretion. Comparable results were also obtained by Kamble et al 2010 in which six fungal isolates secreting cellulase-free xylanase under submerged culture conditions were screened amongst which Rhizopus oryzae (205.0 U ml-1) and Rhizopus microsporus CB-10 (126.0 U ml-1) were the most prominent cellulasefree xylanase producers while Hamigera insecticola (66.0 U mL-1), Rhizopus sp. (75.10 U ml-1), Aspergillus niger (76.0 U ml-1), Aspergillus flavus (48.0 U ml-1), Penicillium citrinum (63.0 U ml-1) and Penicillium sp. (45.0 U ml-1) showed absence of cellulase in submerged fermentation.

To explain the role of different concentration of Ash gourd on the lignocellulolytic enzyme production MEB medium was augmented with 1%, 2%, 3%, 4%, 5%, 6%, 8% and 10% ash gourd and after completion desired incubation period the culture filtrates were collected and enzyme activity was analyzed. The maximal enhancement in the lignocellulolytic enzyme activity was recorded in 5% after which decrease in the enzyme production was observed up to 10% which was still greater than control which was not augmented with ash gourd (Table 2). Graphical representation of enhanced enzyme activity was represented in Fig. 2. Maximum LiP, MnP and laccase activity was revealed when media augmented with 5% ash gourd used in monoculture and co culture. I. lacteus showed an absence of LiP activity throughout the experiment whereas D. confragosa displayed maximum accumulation of LiP in media containing 5% ash gourd concentration after which a decline in the activity was noticed and in co culture of I. lacteus and D. confragosa also 5% ash gourd resulted in the maximum production of LiP enzyme (Fig. 2 A).

In mono culture of I. lacteus MnP activity was enhanced from 0.94 U/L to 1.63 U/L in the range of 1% to 5% concentration of ash gourd after that MnP activity was dropped up to 1. 2 D). The results of xylanase also revealed that 5% ash gourd was the most ideal concentration for xylanase enxyme production after that decline in the xylanase activity was observed up to 10% but it was higher as compared to control. I. lacteus, D. confragosa and co culture of I. lacteus and D. confragosa were producing 34.66, 26.20 and 40.96 U/ml respectively in 5% of ash gourd which was the greatest activity observed amongst all other concentrations (Fig. 2 E). Cellulase enzyme was also seen to be absent even if the media was augmented with ash gourd (Fig. 2 F). The activity of LiP, MnP and laccase, AAO and xylanase enzyme reduced even after the concentration of ash gourd increased in the medium up to 10%. However the enzyme activity showed a decline it was more than the control (ie without ash gourd supplementation). Compared to D. confragosa, I. lacteus showed higher xylanase and AAO enzyme activity and the coculture showed higher enzyme production than monoculture with ash gourd, maximum activity is observed with 5% concentration which is fourfold increase. 10% concentration showed a decline in the activity, but still it is four fold more than the control in which ash gourd is not supplemented to the medium. Ash gourd have higher amount of carbohydrates, specially xylan and pectin (Mazumdar et al 2004, 2005) [25,26] . In the present study Ash gourd pulp was used as a substrate for the first time. In monocultures all the enzymes showed enhancement. When ash gourd was used as a substrate and in co culture LiP and MnP were moderately enhanced but laccase and xylanase were enhanced significantly when the medium was supplemented with ash gourd pulp.

As a outcome of present study to use the Ash gourd pulp for enhancing enzyme activity both the oxidative as well as hydrolytic enzymes enhanced significantly when ash gourd pulp without any other chemical enhancer in monocultures and coculture. Utilization of such low-cost lignocellulosic materials afford an opportunity to obtain high yield of lignocellulolytic enzymes in the simple medium which played major role in the degradation of lignin without augmenting it with expensive enhancers to the culture medium. The selection of lignocellulosic material adequate for the growth of fungus and target enzyme synthesis may lead to develop an efficient technology for scaling up the lignocellulolytic enzyme production.

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