Delignification of Valuable Timbers decayed by India Lignicolous fungi

ABATRACT Wood degrading capacity of lignicolous fungi was studied by decay test. In which two methods were followed, i) wood chips method ii) wood block method. Eight timbers infected by six fungi were selected for studying percentage of decay and biochemical test was done to know delignification. After 12 months, 90 % of wood block of T. arjuna was decayed by L. stereoides. In teak wood 16.82 % of decay was due to H. apiaria in 3 months. As the percentage of moisture was less, percentage of weight loss was also less; this indicated that decay capacity of fungi will depends on % moisture content in wood. The percentage loss in hot water soluble substrates was more in case of T. crenulata due to L. stereoides for 5 months, whereas lowest in case of teak wood decayed by H. apiaria for 5 months. The percentage loss in ethanol benzene soluble substrate was more in case of Adina wood decayed by C. versicolor for 5 months, whereas lowest in case of teak wood infected with L. stereoides for 3 months. As the incubation period increases, percentage loss in acid soluble lignin was more in case of infected woods. L. stereoides, C. versicolor, and H. apiaria showed selective delignification in all infected woods, whereas T. pini showed simultaneous degradation of lignin in all woods tested. The valuable timber like teak wood was not resistant to wood decay because they loss 50% of lignin. The in vitro wood decay test can’t be taken as absolute evidence for wood decay behavior of lignin-degrading fungi, so we should conform decay of wood by consider biochemical test. For rapid evaluation of wood decay the wood chip method was best suitable. For the first time the wood decay and biochemical test of 8 wood samples infected by white rot fungi like S. commune, L. stereoides, H. apiaria, C. versicolor, T. pini and soft rot fungi like T. viride was studied. Histogram 2. The % weight loss of two wood blocks infected with lignicolous fungi. study only 26 % of reduction in lignin was observed in pine and teak wood blocks infected by L. stereoides , 30 % loss in pine and 18 % loss of lignin in teak woods infected by H. apiaria , 31 % loss in teak and 26 % loss of lignin in pine woods infected by T. viride


INTRODUCTION
Wood was a very important byproduct, produced by different biological processes in tree. It was an important natural resource from forests which can contribute greatly to climate change and biodiversity conservation (Kaimowitz D. 2003). Based on the FAO definition there are around 3.7 billion ha of forests in the world. Majority of forest was natural forests, in which more than 50 % in South America and Europe, while plantations cover only 187 million ha, representing 5 % of the total forested area (FAO 2007). Global wood consumption was increasing but at a relatively low pace. In the last 20 years the average global  LTD, 2014 were to 1) survey lesser-known and previously neglected lignicolous fungi for the ability to cause wood decay; 2) elucidate the wood decay and biochemical features of decayed wood by such fungi in comparison with known decay types; 3) decay classification of lignicolous fungi, and; 4) Quick identification of wood decay by different test. In the present paper wood degrading capacity of lignicolous fungi was studied by decay test like wood chip and wood block method. Wood of Tectona grandis, Terminalia arjuna, T. bellerica, T. crenulata, Adina cordifolia, Dalbergia sissoo, Pinus longifolia, and Acacia arabica were selected on the value of timber. Lignicolous fungi like S. commune, L. stereoides, H. apiaria, C. versicolor, T. pini and soft rot fungi like T. viride were used to infect wood chips, blocks and logs of above timbers and these were chemically analyzed.

MATERIALS AND METHODS
Wood degrading capacity of lignicolous fungi was studied by decay test. In which two methods were followed, i) wood chips method ii) wood block method. In biochemical analyses the degraded wood samples were analyzed for water content, pH of samples, solubility in hot water, and ethanol-benzene, acid insoluble lignin and chlorite holocellulose (Dill, I.; Kraepelin, G. 1986).

1. Wooden chips method
A survey was undertaken in forests and sawmills of Gujarat, India, during January 2007 to July 2011, to find out occurrence of lignicolous fungi. These fungi were isolated by PDA medium from sporophore and decayed wood and were grown on 2 % malt extract agar in petriplates for 7d prior to inoculation in decay chambers. Seven woods like T. grandis, T. arjuna, T. bellerica, A. cordifolia, D. sissoo, P. longifolia, and A. arabica wooden chips (0.5g) were added to decay chamber containing Modified Asthana and Hawker's medium 'A'. The composition of medium was 10 g of D -glucose, 3.5 g of KNO 3 , 1.75 g of KH 2 PO 4 , 0.75 g of MgSO 4 ·7H 2 O and 20 g of Agar. The decay chambers were sterilized at 121 ºC for 1 h. The decay chambers were inoculated with Lignicolous fungi like S. commune, L. stereoides. H. apiaria and soft rot fungi like T. viride. Three decay chambers were used for each isolated lignicolous fungi per wood and the fungus inoculated in decay chamber without wooden chips served as control. Assembled decay chambers were incubated in the dark for 20 and 40 d at 27 ±1 ºC. The wooden chips were filtered, oven dried, and weighed. Percent weight loss was determined as follows: Percent weight loss = Weight loss of oven − dried wood after incubation Weight of oven − dried original wood 100 Each set of treatment was run in triplicates and average weight loss was always taken as standard value for comparison of wood decay. The weight loss results were statistically analyzed by using the MS office Excel software and the significant values were taken for study.

Wooden block method
Timber decay caused by Lignicolous fungi like S. commune, L. stereoides. H. apiaria C. versicolor and soft rot fungi like T. viride was observed in wood blocks of T. grandis and T. arjuna and T. bellerica for 20 days to 1year. These fungi were grown on 2 % malt extract agar for 7 d prior to inoculation. The PDA plates were prepared by inoculating each isolate of above fungi and incubated for 7-10 d.
Totally 12 wooden blocks (1 x 1 x 1 cm) per wood per each lignicolous fungi were cut from the respective logs and soaked in distilled water for 30 min. These wooden blocks were autoclaved. After completely spreading of above fungus, four blocks of each wood were placed on the medium per plate and incubated for 3, 6 and 12 months at 27 ±1 ºC. To maintain moisture in test plates two layers of Whatmen filter paper No 1 were placed on the surface of blocks. The sterile distilled water was added to it regularly. The un-inoculated wooden blocks acted as control. After completion of incubation period each block was cleaned (of the mycelium), oven dried and weighed. The percentage weight loses was calculated as described above.

3. Spawn preparation
For spawn preparation a media used for developing sporophores of wood rot fungi as suggested by Etter (1929) was used. The spawn preparation medium consisted of 48 g of corn-meal, 16 g of corn-starch and 8 g of powdered wood. The spawn was taken in a polypropylene bag and 2.5 % malt extract was added. The bag was closed by putting the moist cotton swab to maintain moisture level inside the bag; such bags were sterilized at 121 ºC for 1 h and inoculated with four test fungi like L. stereoides C. versicolor H. apiaria T. pini. The bags were incubated in dark for 15d at 27 ±1 °C. The fully-grown spawn was used for artificial inoculation in wooden logs.

4. Wooden log preparation
The wood log of T. grandis, A. cordifolia, T. crenulata and T. arujna were infected with above lignicolous fungi and used for biochemical analysis. The average size of (4) wood plank used was 2 x 2 x 30 cm length. The bark was not removed. It helps to maintain moisture and keeps away the foreign fungi. A 5/16" drill bit was used to make holes in the logs for insertion of spawn as diamond drilling pattern. After drilling the entire log was autoclaved for 1 h. After autoclaving spawn was inoculated into the holes. It was then sealed off with paraffin wax. Logs were covered with cheese cloth to maintain moisture, packed in polythene bags and incubated in dark for 12 months.

5. Bio -chemical analysis of decayed wood
The chemical composition of sound and decayed wood as determined by previously described technique (Dill, I.; Kraepelin, G.1986). Decayed wood was dried at 105 ºC and then ground to pass through a 60 μ mesh screen. It was used for further analysis.

5. 1. Water content
To obtain water present in the sample decayed wood (3 g) was dried at 105 ºC for about 48 h, cooled in a desiccator and weighed. The difference in two weights gave the water content in milligrams.

5. pH of samples
The pH was determined potentio-metrically after suspension of the samples in distilled water for about 30 to 45 min. Analysis of decayed wood was made with a few grams of fresh material and that of corresponding sound wood was made with 1 g of dry wood meal.

5. 3. Solubility in hot water
One gram of dry wood meal was placed in a 250 ml Erlenmeyer flask. After addition of 100 ml of distilled water, the mixture was slowly stirred at 80 ºC for 3 h. The samples were then filtered by using Whatman filter paper No 1, washed with hot water, dried at 105 ºC for about 24 h, cooled in desiccator and weighed.

5. 4. Solubility in ethanol -benzene
About 1.5 g of dry wood meal was extracted with ethanol -benzene (1:2 v/v) for 4 h in a Soxhlet extractor, keeping the liquid boiling briskly. Each extracted sample was washed with 50 to 100 ml of ethanol and dried at 105ºC. After evaporation of solvent, each extract was dried at 105 ºC for 24 h, cooled in desiccator and weighed.

5. 5. Acid insoluble lignin (klason lignin)
Flasks containing 1 g of ethanol -benzene extracted wood meal and 20 ml of H 2 SO 4 (72 %) were gently shaken in a water bath at 30 °C for 1 h. The acid was then diluted with H 2 O to 4 % (wt/vol), and the samples were autoclaved at 121 °C for 30 min. The lignin that settled overnight was quantitatively collected by filtration through a Whatman filter paper No. 1, washed free of acid with hot water, and dried. The lignin content was calculated as a percentage of oven-dried, non-extracted wood meal.

5. 6. Chlorite holocellulose (CHC)
Chlorite holocellulose was also determined as described by Seifert 91983). Extracted wood samples of approximately 400 mg were placed in 50 ml Erlenmeyer flasks. Seven milliliters of buffer solution consisting of 60 ml glacial acetic acid and 1.3 g sodium hydroxide per 1000 ml distilled water was added to each flask. Three milliliters of 20 % (w/w) aqueous solution of sodium chlorite was immediately added and the flasks were sealed with paraffin wax and aluminum foil. The flasks were placed in an orbital shaker at 110 rpm at 45 °C for 36 to 40 h. after incubation period; flasks were placed in ice bath to stop the reaction. The contents were then transferred to pre weighed Whatman filter paper No. 1 using 100 ml of 1 % acetic acid. The holocellulose was washed with 5 ml of acetone three times and oven dried at 105 °C for 4 to 6 h before weighing.

1. Wooden chips method
Percentage decay of seven different woods caused by S. commune, L. stereoides. H. apiaria (White rot fungi) and T. viride (soft rot fungi) was observed. As compared to other white rot and soft rot fungi, teak and sissoo wood was efficiently degraded by L. stereoides, where the percentage weight loss was 34.6 and 44.6 % after 40days. Whereas in case of T. arjuna, T. bellerica, A. cordifolia, A. arabica, and P. longifolia woods were efficiently  The molecular structure of wood suggests that cellulose a structural component thereof could be bio-recycled into glucose, a fermentable sugar (Reddy N. and Yang Y. 2009). Cellulase, a multi-component enzyme system produced by soft rot fungi such as T. viride and Aspergillus niger exhibits the ability to saccharify cellulose. These enzymes have been proved to be effective in the bioconversion of wood products such as wastepaper into fermentable sugars (van Wyk J. P. H. 2001). In the present paper the ability of soft rot fungi to degrade lignin was proved by wood chip test. The various sawdust wood samples were exposed to T. viride cellulase action with the delignified cellulose component bio-converted into fermentable sugars such as glucose (Bohdan V, Yaser D 2011). In the present study the T. viride showed significant weight loss in seven woods so it may be used for saccharify cellulose.

2. Wooden block method
Wood decay caused by four test organisms was observed in wood blocks of T. grandis and T. arjuna wood blocks after every 20, 40 and 60 days. The maximum decay was shown by L. stereoides in case of T. arjuna after 60 days. The minimum decay was observed in case of T. arjuna due to S. commune after 20 days. In initial stages of decay, percentage of 108 ILNS Volume 16 moisture was more whereas in advanced stages of decay the % moisture reduced. As the percentage moisture was less the percentage weight loss was also less, this indicates that the decay capacity of lignicolous fungi depends on the % moisture content in wood (Table 2 and Histogram 2).

International Letters of Natural Sciences Vol. 16
Percentage wood decay of three different woods T. grandis T. bellerica and T. arjuna was infected by white rot fungi like L. stereoides, C. versicolor and H. apiaria for 3, 6, 12 months. After 12 months 90 % of wood was decayed in T. arjuna due to L. stereoides, followed by T. bellerica and T. grandis. Wood decay was minimum (16.82 %) in teak due to H. apiaria after 3 months of inoculation (Table 3 and Histogram 3).

ILNS Volume 16
Biological agar block method allowed wood samples to be evaluated and monitored in terms of colonization and development of decay by C. versicolor and classified based on mean mass loss. In this research, the in vitro decay of five commercial woods by C. versicolor was studied by the agar block method. The selected wood samples were Abies alba. Populus alba, Fagus orientalis, Platanus orientalis and Ulmus glabra. There was a high correlation between the mass loss and apparent damage. Therefore biological evaluation of wood regarding biodegradation and selection of wood types for various applications will be of high priority (Olfat A.M., Karimi A.N., and Parsapajouh D. 2007). In the present study also there was a correlation between the weight loss and damage, so different biochemical test were studied to conform that biological evaluation of wood was necessary.
The Basidiomycetes, Poria carbonica and C. versicolor, caused substantial wood weight loss over the test period. These results were usually obtained by soil or vermiculite burial methods (Morrell J.J. and Zabel R. A. 1989). In the present study the wood chip and block method showed significant weight loss in seven woods infected by white rot and soft rot fungi. The decreasing weight in studied samples showed that C. versicolor can grow quickly and may rapidly affect the appearance and degrade the wood (Olfat A. M., and Karimi A.N., 2005). In the present study the growth of C. versicolor was quick to degrade the wood very fast when compared to other lignicolous fungi. The lowest weight loss decreasing was observed in U. glabra and highest value in F. orientalis. This was true for the study of crude oil and beech wood caused by C. versicolor (Olfat A. M., and Karimi A.N., 2005). In the present study also C. versicolor degrade T. arjuna wood very fast.
The percentage weight loss in inner heart wood and outer heart woods of New guinea teak was 44, 54 and 12, 21 for Coniophora olivacea and C. versicolor respectively. The percentage weight loss in inner heart wood and outer heart wood of Indonesian teak was 54, 55 and 22, 21 for C. olivacea and C. versicolor. The percentage weight loss of inner heart wood and outer heart wood of Burma teak was 4, 8 and 4, 3 for C. olivacea and C. versicolor respectively (Guilley et al. 2004). In the present study the heart wood of teak showed 72.68 %, 54.15 %, 64.88 % of weight loss by L. sterioides, C. versicolor and H. apiaria respectively. Based on percentage weight loss, the American Society for Testing Materials ASTM (1969) classified resistance of wood. Highly resistant wood showed weight loss of zero to 10 %, resistant wood shows weight loss of 11 to 24 %, moderately resistant wood showed 25 to 44 % weight loss, and nonresistant wood showed 45 % or greater weight loss. In the present study the teak and terminalia wood was moderately resistant to non resistant when infected with different lignicolous fungi in wood block test. The in vitro decay of five commercial woods by C. versicolor was studied by the agar block method showed strong resistance of U. glabra and lowest resistance in F. orientalis (Olfat A.M., Karimi A.N., and Parsapajouh D. 2007). In the present study teak wood infected with C. versicolor showed resistant to non resistant. Where as in terminalia wood infected with same fungi showed moderately resistant to nonresistant. Twelve hundred samples from 31 trees were exposed to four fungi: Pycnoporus sanguineus, Antrodia sp., Gloephylum trabeum, and Coriolus versicolor. Tests showed that Antrodia sp. and C. versicolor resulted in <20 % mass loss, whereas all samples were rated as durable or highly durable with regard to P. sanguineus and G. trabeum. Inner heartwood was found to be the most resistant to pathogen attack and outer heartwood the least (Kokutse, et al. 2006). In the present study teak wood infected with L. stereoides showed moderately resistant to nonresistant, with C. versicolor and H. apiaria showed resistant to non resistant.
So, the in vitro wood decay test cannot be taken as absolute evidence for the behavior of lignicolous fungi, they were useful to determine their wood-degrading properties. Weight International Letters of Natural Sciences Vol. 16 loss of yellow-poplar samples incubated with T. versicolor in a soil-block test was significantly higher than that in an agar-block test. Therefore, the soil-block test was more sensitive to detect fungal decay in yellow-poplar under the conditions of the experiments (Schirp A. and Wolcott M. P. 2005). In the present study the most effective method was agar block method when compared to wood chip method.

1. Artificially inoculated wood blocks
The physicochemical analysis of teak, and pine woods infected with wood decay fungi was done. The details were recorded in Table 4 and 5.  Highest percentage of moisture was shown by teak wood infected with H. apiaria whereas it was lowest in case of pine wood infected with S. commune. High acidic nature was shown by teak wood infected with H. apiaria in 20 days. Almost neutral nature was shown by teak wood infected with T. viride. The percentage loss of ethanol -benzene soluble substrates was more in case of teak wood decayed by H. apiaria for 40 days, whereas, lowest was observed in pine wood decayed by L. stereoides1 and T. viride. The highest percentage loss of acid insoluble lignin was observed in case of teak wood decayed by H. apiaria for 40 days, whereas, lowest in case of teak wood inoculated with L. stereoides 2. Where as highest percentage loss of Acid insoluble lignin was observed incase of pine woods decayed by H. apiaria and lowest incase of pine wood inoculated with L. stereoides 2. The percentage loss of holocellulose was more in case of teak wood infected with L. stereoides 2, whereas, lowest in case of wood decayed by H. apiaria. The highest loss of holocellulose in pine wood was observed by H. apiaria decay and lowest in case of S. commune decay Three white rot fungi Daedalea elegans, Polyporus glaganetus, and L. betulina were screened for their lignin degrading abilities on rice straw, maize cob, sawdust of Terminalia superba and sugarcane bagasse at different time intervals (30,  When a brown-rot-causing fungi Polyporus palustris was infected to Mangifera indica wood shavings for considerable periods, approximately 40 to 50 % lignin loss was observed in two years (Ananthanarayanan, S.; Wajid, S.A.; Padmanabhan, S. 1978). The Mangifera wood blocks were infected with white-rot-causing fungi for 90 days, the utilization of lignin was 26 % by F. flavus and 20 % by S. commune (Padhiar A., Albert S., Nagadesi P. K. and

CONCLUSIONS
The wood degrading capacity of lignicolous fungi was studied by decay test. In which two methods were followed, i) wood chips method ii) wood block method. For rapid detection of decay in wood, the wood chips method was best. When we compare Soil Block method, Agar Block method and wood chip method, the agar block method was best to study decay pattern and Biochemical changes in wood. As the fungi required suitable conditions like suitable temperature, moisture, pH for better growth and also for decay of wood. In the present paper in initial stages of decay the percentage of moisture was more, whereas in advanced stages of decay the % moisture was less. As the percentage moisture was less percentage weight loss was also less, this indicates that the decay capacity of fungi depend on % moisture content in wood. Based on the weight loss studies different scientists have explained that the woods were resistant to a particular fungi. But in present study it was found that in case of lesser weight loss also wood was severely degraded by the lignicolous fungi. Therefore, on the basis of weight loss studies alone the type of wood decay can not be certainly decided. As the incubation period increased the percentage loss in acid soluble lignin was more in case of all infected woods. L. stereoides, C. versicolor, and H. apiaria showed the selective delignification in all infected woods, whereas, T. pini showed simultaneous degradation of lignin in all woods tested. The valuable timber like teak wood was not resistant to wood decay by lignicolous fungi used because they utilized 50% of lignin. For the second time the ability of wood decay by soft rot fungi like T. viride was described. For the first time the biochemical changes like loss of klason lignin and holocellulose from T. viride infected woods was described. For the first time the wood decay and biochemical changes in T. crenulata, T. bellerica, and T. arjuna woods infected by L. sterioides, C. versicolor H. apiaria T. pini, S. commune, and T. viride was described.