Comparative ligninolytic potential of Phlebia species and their role in improvement of in vitro digestibility of wheat straw

Digestibility of crop-residues can be enhanced by the delignifi cation, which improves its nutritive quality. Presence of lignin in these crop-residues which act as digestibility barrier can be degraded by fungi. The biodelignifi cation ability of three Phlebia species: Phlebia radiata, Phlebia fascicularia and Phlebia brevispora, was studied under solid state fermentation of wheat straw for 30 days and compared with that of much studied white rot fungus Ceriporiopsis subvermispora. Different parameters including laccase activity, loss in total organic matter, water soluble content, hemicellulose, cellulose, lignin, ash content, change in pH and effect of the chitin content of fungus on in vitro digestibility were examined before and after the fungal treatment of substrate. P. brevispora caused maximum lignin loss of 305 g/kg with the lowest loss in total organic matter (105 g/kg) and enhanced the digestibility to its maximum extent (259 g/kg). C. subvermispora which degraded 230 g/kg lignin gave maximum in vitro digestibility of 221 g/kg only.


INTRODUCTION
Several crop-residues like wheat straw, rice straw, maize stover etc., are generally used as substitutive fodder as well as its supplement.These residues can be converted into high quality feed by means of fungal delignifi cation, which improves its nutritive value (Zadrazil and Brunnert, 1980;Reid, 1989;Vills-Boas et al., 2002).Natural degradation of plant residue is mainly caused by different fungal and bacterial species.Holocellulose and lignin are the biopolymers that are converted into low molecular weight compounds by the enzymatic action of these microorganisms.Fungi are usually better degraders of plant cell wall constituents because of their hyphal penetration power.Many fungal species have been screened for their potential to degrade cereal residues.White rot fungi are the most studied and degrade lignin effi ciently by using their ligninolytic enzyme system (Agosin et al., 1985;Arora et al., 2002).Lignin is bound to cellulose and hemicellulose and makes these components inaccessible for microbial attack.Animal enzymatic system does not support ligninolysis and so these crop-residues are not completely digestible and thus of poor nutritive value.Fungal ligninolysis and break down of cellulose-hemicellulose matrix liberate simple degradable components that can be easily utilized by rumen microfl ora, thus improving the ruminant digestibility.
Phanerocheate chrysosporium is a well-known white rot fungus and most widely studied for lignin degradation.However, loss of total organic matter is very high during the degradation of lignocellulosics, which limits its practical use and necessitates looking towards selective ligninolytic organisms (Jung et al., 1992).Phlebia species are known selective lignin degraders (Arora and Gill, 2000) and thus may possess the potential capability for improvement of digestibility of agro-residues.Four white rot fungi including three different species of Phlebia and another widely studied fungus Ceriporiopsis subvermispora (Akin et al., 1995) were selected to fi nd out their comparative role in selective ligninolysis and in vitro digestibility of wheat straw.In vitro digestibility was measured by using buffalo faecal matter rather than ruminant fl uid as a substitutive source of microfl ora.This is relatively easy method than Tilley and Terry's (1963) two-stage in vitro procedure, which requires special surgical skills, fi stulated animal, where special care is needed to keep fi stula free from any infection.On the other hand, the method involving the use of enzymes, like cellulase, is relatively expensive (Nousiainen et al., 2003).Use of faecal inoculum for determining the digestibility is comparatively cheaper and easier method for practical use in a microbiological laboratory (Shaer et al., 1987;Akhter et al., 1999).

Experimental procedure
Wheat straw (WS) used as a substrate was milled (particle size 2 mm ± 0.5), washed (with water at 25-30ºC) and dried at 90ºC.Five g of dried WS taken in 250 ml conical fl asks was moistened with 25 ml of 0.5% (w/v) malt extract and after sterilization at 1.06 kg/cm 2 for 15 min, the fl asks were inoculated with three mycelial discs (8 mm) per fl ask, grown on YGA plates for 6 days.All the fl asks were incubated at 25ºC.Two fl asks for each organism were processed at 10, 20, and 30 days along with 2 uninoculated control fl asks.The whole experiment was repeated three times.Thus, the data presented in Tables 1 and 2 is an average of six individual fl asks.Enzyme extraction was done as described earlier (Arora et al., 2002).Twenty fi ve ml of sodium acetate buffer (10 mM, pH 5) was added to each fl ask and put on a rotary shaker at 200 rpm for 20 min.The contents of each fl ask were fi ltered on a tared fi lter paper and dried at 90ºC until constant weight.Filtrate obtained was centrifuged at 8000 g at 4ºC for 15 min and the extract thus obtained was used for laccase assay.The dried residue was used for different analytical tests.

Analytical methods
The sequential fractionation of lignocellulosics was carried out according to Datta (1981) with slight modifi cations.One g of WS was suspended in 100 ml distilled water, kept at 100°C for 2 h in a water bath and fi ltered on a tare crucible, residue was dried at 90°C till constant weight.Loss was considered as water soluble part.Dried residue was suspended in 100 ml of 0.5 M H 2 SO 4 and after keeping for 2 h at 100°C in a water bath, the contents were fi ltered, dried and weighed as described in the fi rst step and loss in weight is represented as hemicellulose content.For cellulose and lignin estimations, 10 ml of 72% (v/v) H 2 SO 4 was added to the above dried residue and kept at 30°C for 1 h on a rotary shaker at 200 rpm.After incubation the mixture was diluted up to 4% (v/v) of H 2 SO 4 and autoclaved at 1.06 kg/cm 2 for 40 min.The contents were fi ltered, dried and weighed.The loss in weight was treated as cellulose, and the left over residue was considered as lignin.
For estimating the residual ash content, 1 g of sample was kept at 550ºC for 5 h in a tare crucible and reweighed to calculate the residual ash content.Change in pH of the decomposed and undecomposed WS was determined by taking for 30 min.pH was read with the help of pH meter.

Laccase assay
Laccase (EC 1.10.3.2) activity was measured as described earlier (Arora and Gill, 2005).Five ml of reaction mixture containing 10 mM sodium acetate buffer (pH 5), 2 mM guaiacol and 0.2 ml of culture supernatant was incubated at 25ºC for 2 h and the absorbance was read at 450 nm.The relative enzyme activity has been expressed as colorimetric units/ml (CU/ml).

Chitin estimation
Chitin was measured according to Chen and Johnson (1983).All the fungi were grown in 50 ml yeast extract glucose broth for 15 days at 25ºC.The fungal biomass so obtained was dried at 90ºC for 24 h.Ten mg of dried fungal biomass was refl uxed at 100°C for 4 h in 5 ml of 6 M hydrochloric acid.After cooling to room temperature, hydrolysates were fi ltered and 1 ml aliquots of such samples were withdrawn and evaporated to dryness at 60°C under reduced pressure.The dry hydrolysates were then redissolved in 4 ml distilled water.To 1 ml of this diluted solution, 0.25 ml of acetylacetone (4% acetylacetone in 2.5 M sodium carbonate) was added and heated at 90°C for 1 h in a capped test tube.After cooling, 2 ml of ethanol and then 0.25 ml of Ehrlich reagent (1.6 g of N-N-dimethyl-p-aminobenzaldehyde in a 30:30 ml mixture of ethanol and concentrated HCI) was added.The colour formed was measured at 530 nm.The glucosamine hydrochloride content of cell wall hydrolysates was determined by comparing its absorbance with that of standard glucosamine hydrochloride.

In vitro digestibility
In vitro digestibility of uninoculated and fungal treated WS was estimated according to Akhter et al. (1999), with slight modifi cations.Faecal inoculum was prepared by mixing fresh faecal matter of buffalo (100 g/l) in pre-warmed (39ºC) artifi cial saliva (NaHCO 3 9.80 g, Na 2 HPO 4 .7H 2 O 7.00 g, KCl 0.57 g, NaCl 0.47 g, MgSO 4 .7H 2 O 0.12 g and 1 ml CaCl 2 (4%, w/v) in 1000 ml of distilled water) and fi ltered through four-layered muslin cloth.Five hundred mg WS (washed and dried) taken in 50 ml centrifuge tube was suspended in 35 ml of faecal inoculum.After fl ushing with CO 2 gas, these tubes were kept at 39ºC for 48 h in a water bath.Supernatant was discarded and 35 ml of acidifi ed pepsin (6.6 g in 1 l of 0.1 M HCl) was added to the residue.Tubes were again incubated at the same conditions for 48 h.Residue was fi ltered on a tared fi lter paper and dried.The weight loss in dry matter during the incubation has been expressed as in vitro digestibility.

Statistical analysis
The data was represented as mean with standard deviation and analysed by two-way ANOVA (4 X 3 factorial design) and correlation.

RESULTS
Solid-state fermentation of wheat straw was carried out with different white rot fungi and compared with that of the properties of uninoculated WS.Uninoculated WS contains 85 g/kg water solubles, 353 g/kg hemicellulose, 325 g/kg cellulose, 240 g/kg lignin and 70 g/kg residual ash with the in vitro digestibility of 143 g/kg and pH 6.04.The fungi were examined for their potential to degrade different plant cell wall components and enhancement in in vitro digestibility during 30 days of incubation as stationary cultures at 25ºC (Table 1).
All the fungi caused variable loss in total organic matter (TOM) during solid state fermentation of WS.P. radiata caused the maximum loss (159 g/kg) during 30 days of incubation.P. fascicularia, P. brevispora and C. subvermispora did not show any statistically signifi cant difference in the resultant loss in TOM, which ranged from 100-108 g/kg (Table 1).Different fungi were consistent in their rate of degradation during 30 days period except P. fascicularia, which caused more loss during 10-20 days of incubation.
A maximum of 127 g/kg water soluble components were liberated by C. subvermispora during 20 days while P. brevispora and P. fascicularia liberated maximum water solubles during 30 days (120 and 98 g/kg, respectively) and P. radiata (97 g/kg) during 10 days.C. subvermispora was more effective in the early period of incubation and released more water solubles during fi rst 20 days with very little increase during further incubation while Phlebia species were more active in releasing water solubles in 20-30 days period (Table 1).
P. fascicularia was the best degrader of cellulose and caused a maximum loss of 263 g/kg, followed by P. brevispora and C. subvermispora by causing around 190 g/kg loss, whereas P. radiata caused a loss of 173 g/kg in 30 days.P. radiata, P. brevispora and C. subvermispora were more active during 10-20   days in cellulose degradation as compared to P. fascicularia, which degraded cellulose more in last 10 days.All the fungi were able to degrade lignin; P. brevispora was the best lignin degrader and resulted in maximum lignin loss of 305 g/kg in 30 days of incubation.It was closely followed by P. radiata (289 g/kg), whereas C. subvermispora and P. fascicularia caused a lignin loss of 230 and 221 g/kg, respectively.P. radiata and C. subvermispora were relatively faster degraders of lignin and resulted in 170 and 157 g/kg loss in fi rst 10 days of incubation, while P. brevispora was better in later stage of incubation, i.e. 20-30 days (Table 1).
In all the fungal treated substrates, pH declined sharply during fi rst 10 days of incubation and fell to 3.5-5.4as compared to uninoculated WS (pH 6.04).However, it increased slightly thereafter (Table 2).Residual ash content of degraded WS increased with increase in incubation period and maximum resultant ash (in 30 days) was obtained from WS degraded by C. subvermispora (102 g/ kg), which was followed by P. radiata (95 g/kg), P. brevispora (94 g/kg) and P. fascicularia (85 g/kg).The residual ash content resulting from the degradation of WS by different fungi was statistically signifi cant (P<0.05)except P. radiata and P. brevispora.WS treated with P. brevispora showed the highest in vitro digestibility of 259 g/kg followed by P. radiata (232 g/kg), P. fascicularia (231 g/kg) and C. subvermispora (221 g/kg) during 30 days, whereas the control WS showed 143 g/kg digestibility.Difference in in vitro digestibility resulting from the WS treated with different fungi was statistically signifi cant (P<0.05)except P. radiata and P. fascicularia.After fi rst 10 days, only P. brevispora was able to enhance the digestibility up to a signifi cant level which continued during the entire incubation period while other fungi enhanced digestibility during 10-30 days of incubation.
Different fungi showed different laccase production profi le.P. brevispora was the best laccase producer and resulted in a maximum laccase production during 20 days, which was followed by P. fascicularia during 30 days, C. subvermispora during 10 days while laccase production by P. radiata was minimum as compared to other fungi (Figure 1).

DISCUSSION
Chemistry of lignin confers resistance to wood and other agricultural residues against microbial attack.However, white rot fungi have got the necessary potential to degrade lignin because of their well defi ned ligninolytic enzyme system, which make them suitable candidates for delignifi cation of forage crops to improve their digestibility (Zadrazil, 1977).Invariably these fungi also degrade cellulose and hemicellulose along with lignin.The loss of these components from the feed is not economically desirable because less biomass is available for animal feed.In an earlier study Phanerocheate chrysosporium, a common and widely studied fungus, degraded lignin but also caused a high loss in total organic matter (TOM) (Jung et al., 1992).To overcome this problem, selective ligninolysis needs to be carried out.In the present studies, all the white rot fungi promise to be a good choice of being selective lignin degraders with minimum loss in TOM.
From the experimental data, the lignin loss as caused by different fungi can be correlated with the enhancement of digestibility of substrate (Correlation coeffi cient = 0.915, which shows a very strong positive correlation).In vitro digestibility of wheat straw was maximum in P. brevispora, which caused maximum lignin loss and minimum loss in TOM.All the fungi were selective in lignin degradation and enhanced the in vitro digestibility accordingly.It also supported the earlier observations of Mukherjee and Nandi (2004) during their study on two Pleurotus species.After 20 days of incubation P. brevispora enhanced the in vitro digestibility up to 220 g/kg with 61 g/kg loss in TOM and there was no statistically signifi cant difference when compared with enhancement in the in vitro digestibility caused by C. subvermispora after 30 days of incubation with a simultaneous loss of 108 g/kg in TOM (Table 1).Thus, the use of P. brevispora is practically benefi cial for improving the quality of wheat straw.Next to P. brevispora, P. radiata and P. fascicularia gave almost similar results.P. radiata degraded more lignin than P. fascicularia but there was no statistically signifi cant difference in the in vitro digestibility of substrate treated by these fungi.Both the Phlebia species had same impact on the digestibility improvement, but economically P. fascicularia may prove to be more suitable candidate because of minimum loss in TOM and faster rate of digestibility enhancement.Martínez et al. (2005) reported that lignin and hemicellulose were attacked initially while the cellulose degradation rate increased later, which is in consonance with the present study.Initially all the fungi consumed simple components which did not give any signifi cant difference in digestibility except P. brevispora.Concentration of water soluble components depends upon the degradation of complex compounds like cellulose and hemicellulose, which are converted into lower molecular weight components.However, water solubles are not directly related to digestibility though may be signifi cant nutritionally.P. radiata released maximum water solubles but it was unable to give a proportional enhancement of in vitro digestibility in consonance with earlier fi ndings of Rolz et al. (1986) with lemon grass and citronella bagasse as substrate and 12 white rot fungi.
In line with maximum ligninolysis, P. brevispora gave the highest laccase yield followed by P. fascicularia and C. subvermispora.Though, laccase activity was minimum in P. radiata but it degraded the lignin in a very selective and effi cient manner, which shows that the lignin degradation also depends upon other ligninolytic enzymes, i.e. mangnese peroxidase and lignin peroxidase, etc. (Arora et al., 2002).
Chitin is a basic structural component of fungal cell wall.Presence of fungal mycelia in these plant cells thus increase the chitin content, which may not be easily digested by ruminants.Amount of chitin depends upon the fungal species and its growing conditions.Thus, the chitin content may affect the digestibility of feed.Chitin may not be of much nutritional value to ruminants but its content can affect the digestibility of the fungal treated substrate.Maximum chitin was estimated in C. subvermispora, which showed the lowest increase in digestibility, while the fungus P. brevispora having the lowest chitin content enhanced the in vitro digestibility to its maximum extent, which is in the consonance with the study of Asiegbu et al. (1994).Other fungi also showed the relation between their chitin content and digestibility.P. fl oridensis also enhances the digestibility of WS up to 250 g/kg with a minimum loss of TOM, this fungus also has a lower amount of chitin (data not shown).C. subvermispora and P. radiata also degraded lignin selectively making them suitable choice with specifi c reference to pulping industry (Yaghoubi et al., 2008) while P. brevispora may turn out to be better fungus for practical use to improve the digestibility of wheat straw.

Figure 1 .
Figure 1.Laccase production by different white rot fungi during solid state fermentation of wheat straw for 30 days

Table 1 .
Solid state fermentation of wheat straw and changes in its biochemical composition and digestibility by four different fungi Item

Table 2 .
Changes in pH profi le of WS during its degradation by different fungi