Microorganisms
Two potential ligninolytic fungi such as Mucor circinelloides GL1 (GenBank Accession No.: MF458974) and Fusarium verticillioides GL5 (GenBank Accession No.: MF458977) which were indigenous to the litter samples of Western Ghats of Karnataka (India) were selected (Unpublished data) and used to study their efficiency in decomposing some agricultural wastes in the laboratory.
Agricultural wastes
Three different agricultural wastes such as areca husk, coffee husk and paddy straw were used as the solid substrates to determine the efficiency of selected potential ligninolytic fungal strains to decompose them. The solid substrates were procured from the local agricultural farms of Kodagu district in Karnataka (India) during January, 2017. The substrates were brought to the research laboratory and oven dried at 80 °C. The dried substrates were pulverized and sieved through 20 mesh sieve size and then stored at room temperature in the polyethylene bags for further experiments.
Solid-state fermentation process of agricultural wastes by ligninolytic fungi
Solid-state fermentation process was performed to check the different substrates degradation efficiency of selected ligninolytic fungal strains (Takó et al. 2015). Briefly, 10 g dried substrate was moistened with 40 mL sterile water in 250-mL Erlenmeyer flask and autoclaved for 20 min at 121 °C. Five mycelial plugs (5 mm of diameter) of 7-day-old fungal culture actively growing on malt-extract agar (MEA) medium (2%) were inoculated onto the flasks separately containing three different sterilized solid substrates. The flasks separately containing different sterilized solid substrates without fungal inoculation were used as the controls. The flasks were then incubated under static conditions in dark at 28 ± 2 °C for 120 days. For better fungal growth, the moisture content was maintained at 70% throughout the incubation period of decomposition by sprinkling the sterile water regularly in each experiment. The decomposed samples were harvested at the periodic intervals of 0, 20, 40, 60, 80, 100 and 120 days after fungal inoculation and used for determining the various parameters as described below. After harvesting, the decomposed samples were washed twice with sterile water to remove the mycelia, dried and then used for further experiments.
Physico-chemical analyses
Determination of pH
The decomposed substrate was homogenized with deionized water at the ratio of 1:10 (w/v) by shaking gently for 30 min at 200 rpm. The mixture was filtered through a Whatman filter paper and centrifuged for 10 min at 6000 rpm. The pH of filtrate was recorded using a digital pH meter.
Determination of total organic matter and organic carbon content
The total organic matter of the decomposed substrate was assessed by using the loss on ignition (LOI) method of Salehi et al. (2011). The decomposed substrate was heated to 560 °C by placing it in a ceramic crucible until constant weight was achieved in the muffle furnace. Then, the dried sample was cooled in the desiccator and weighed. Total organic matter was calculated by using the following formula.
$${\hbox{Total organic matter (mg/g)}}=\frac{\hbox{Initial weight of sample }-{\hbox{ Final weight of dried sample}}}{\hbox{Initial weight of sample}}$$
The total organic carbon of the decomposed substrate was then calculated by dividing the total organic matter by the factor of 1.724.
$${\hbox{Total organic carbon (mg/g)}}=\frac{\hbox{Total organic matter}}{1.724}$$
where 1.724 is the factor for converting the total organic matter to total organic carbon.
Determination of total nitrogen and crude protein
The total nitrogen of the decomposed substrate was analyzed by using the micro-Kjeldahl acid digestion method of Rhee (2001). The decomposed substrate was digested with concentrated sulfuric acid and distilled into 4% boric acid solution using the micro-Kjeldahl distillation apparatus. Then, the aliquot was titrated against diluted sulfuric acid with the blank. The total nitrogen was calculated from the volume of sulfuric acid consumption using the following formula.
$${\hbox{Total nitrogen content}}\,\left( {\hbox{mg/g}} \right) = \frac{{{\hbox{Titer value}}\, \times \,{\hbox{N of H}}_{2} {\hbox{SO}}_{4} \, \times \,14.007}}{{{ }1000\, \times \,{\hbox{Weight of the sample in g}}}}$$
where the titer value is the difference between the volume of consumption of sulfuric acid in the test sample and blank, 14.007 is the molecular weight of nitrogen (g/mol), and 1000 is the conversion factor (mL to L).
The crude protein of each substrate was calculated by using the following formula.
$${\hbox{Crude protein content (mg/g)}}={\hbox{Total nitrogen content}}\times 6.25$$
Determination of total phosphorus
The total phosphorus of the decomposed substrate was estimated by using the molybdenum-blue spectrophotometric method after ascorbic acid reduction (Singh et al. 2015). The decomposed substrate was incinerated at 550 °C for about 3 h in muffle furnace, powdered and digested with the mixture of tri-acids (sulfuric acid, perchloric acid and nitric acid) at 300 °C. After digestion, the digest was filtered through a Whatman filter paper and reacted with fresh mixed reagent (ammonium molybdate, antimony potassium tartrate and 50% sulfuric acid). The reaction mixture was reduced by the acidified ascorbic acid solution (as a reducing agent) to form the molybdenum-blue colored complex. The blue color intensity was measured at 880 nm using spectrophotometer. The total phosphorus content was estimated by using the standard curve plotted with the absorbance against the known concentration of standard phosphorous. The content was expressed as mg/g of the decomposed substrate.
Determination of total potassium
The total potassium of the decomposed substrate was estimated by using the flame emission spectrophotometric method (Néel et al. 2014). The decomposed substrate was incinerated at 550 °C for about 3 h in muffle furnace, powdered and digested with the mixture of tri-acids (sulfuric acid, perchloric acid and nitric acid) at 300 °C. After digestion, the digest was extracted with deionized water and filtered through a Whatman filter paper. Then, the filtrate was fed into the flame emission spectrophotometer and read the absorbance at 766 nm. The total potassium was calculated by using the standard curve plotted with the absorbance against the known concentration of standard potassium. The content was expressed as mg/g of the decomposed substrate.
Determination of carbon to nutrient supply (N, P and K) ratio
Carbon to nutrient supply (N, P and K) ratio of the decomposed substrate was calculated by dividing the total organic carbon content with each nutrient (such as total nitrogen, total phosphorus and total potassium).
Determination of lignocelluloses content and loss
Determination of lignin content and loss
The lignin content before and after fungal treatment was evaluated by measuring Klason lignin and acid soluble lignin of the substrate. The gravimetric measurement of Klason lignin content of the substrate was performed as per the modified Klason method using hot sulfuric acid digestion (Fagerstedt et al. 2015). Briefly, the substrate was treated successfully with sulfuric acid (72% v/v) to depolymerize the crude cellulose and hemicellulose for 1 h with stirring frequently to assure the complete solution at 30 °C. The acid was then diluted with deionized water to make 3% (v/v) sulfuric acid for the hydrolysis of dissolved polysaccharides. The suspension was subsequently autoclaved for about 1 h at 121 °C and cooled to room temperature. The precipitate was filtered and dried for 4 h at 525 °C in the muffle furnace. After cooling, the dried sample was weighed to determine the Klason lignin content, acid insoluble residue and ash content present. Then, Klason lignin is calculated by subtracting the acid insoluble ash from the acid insoluble residue. The acid soluble lignin was estimated by reading the absorbance spectrophotometrically at 205 nm using the absorption coefficient (ε = 110 M−1 cm−1). The total lignin content was determined by the addition of Klason lignin with the acid soluble lignin. The change in lignin content was measured to calculate % of lignin loss by using the following formula.
$${\hbox{Lignin loss}}\,({\%})=\frac{\hbox{Initial lignin content }-\hbox{ Final lignin content}}{\hbox{Initial lignin content}} \times 100$$
Determination of holocellulose content and loss
The holocellulose (i.e., the total carbohydrate fraction containing both cellulose and hemicellulose) content of the substrate before and after fungal treatment was determined (Rabemanolontsoa and Saka 2012). The sample was ground and extracted with the mixture of alcohol:benzene (1:1 v/v) at 70 °C for 8 h to remove the extractives. One gram of the sample was suspended in 150 mL of sterile water. The solution was mechanically stirred. Ten drops of glacial acetic acid and sodium chlorite (1.5 g) were added to the solution with vigorous shaking. The mixture was then incubated in a water bath for about 1 h at 70 °C. After incubation, the same quantity of reagent was added and the same process was repeated until the total reaction time was 4 h. The solution was cooled to room temperature, filtered and washed with sterile water until free of acid, then followed by washing with acetone. The residue was oven dried at 105 °C for 4 h, cooled in the desiccator and weighed. Holocellulose content was calculated by using the following formula.
$${\hbox{Holocellulose content }}\,({\%})=\frac{\hbox{Oven dried weight of holocellulose}}{\hbox{Oven dried weight of initial sample}} \times 100$$
The change in holocellulose content was measured to calculate % of holocellulose loss by using the following formula.
$${\hbox{Holocellulose loss }}\,({\%})=\frac{\hbox{Initial holocellulose content }-\hbox{ Final holocellulose content}}{\hbox{Initial holocellulose content}} \times 100$$
Enzyme activity
Extraction of enzymes
The enzymes were extracted from fermented and unfermented solid substrates according to the procedure of Leite et al. (2019) with slight modification. The decomposed substrate (10 g) was suspended in 50 mL of sodium acetate buffer (50 mM pH 5.0) and gently shaken for 30 min at 150 rpm. The content was transferred to a muslin cloth and filtered. The supernatant was then centrifuged at 10,000 rpm for 15 min at 4 °C. The resultant filtrate was served as a source of crude enzyme for the estimation of protein content and the activity of ligninolytic enzymes and carboxymethyl cellulase.
Estimation of protein content
The protein content was estimated at the wavelength of 595 nm by Bradford method (He 2011) using bovine serum albumin (BSA) as a standard. The protein content was expressed as mg/mL.
Estimation of activity of enzymes
The activity of ligninolytic enzymes [laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP)] and carboxymethyl cellulase (CMCase) was determined. Laccase activity was determined by monitoring the oxidation of guaiacol as per the method described by Arora and Sandhu (1985). The MnP activity was determined by monitoring the oxidation of phenol red as per the method described by Orth et al. (1993). The LiP activity was determined by monitoring the oxidation of dye azure B as per the methods described by Archibald (1992), and Arora and Gill (2001). The CMCase activity was determined as per the method described by Miller (1959) using 1% (w/v) carboxymethyl cellulose (CMC) as a substrate and 3,5-dinitrosalicylic acid (DNS) as a coupling reagent. The enzyme activity was expressed as International Units (IU) per mL.
Statistical analysis
For each solid substrate, all the assays were performed by using the randomized complete block design (RCBD) with triplicates. Each experiment was repeated three times. The experimental data were analyzed statistically and subjected to one-way ANOVA by using IBM SPSS Statistics, version 23 (Wagner 2016). The significant differences observed between the treatment means were determined by the highest significant difference which was calculated by using Tukey’s test at level p ≤ 0.05.