Biodegradation of different genotypes of Miscanthus by wood rot fungi

Abstract

Miscanthus, which is comprised of several different genotypes, is an important high-biomass crop with applications in the biofuel industry and in the formation of biocomposite materials. The overall composition of Miscanthus can be altered via degradation with wood rot fungi. The starting composition revealed that the cellulose content of Miscanthus x giganteus was higher than that in Miscanthus sacchariflorus and that the lignin contents were similar in both genotypes. Of the wood rot fungi, only Lentinus edodes appeared to have completely colonized M. sacchariflorus and showed significant degradation. In contrast, all of the brown rot fungi showed partial colonization of both Miscanthus genotypes and had little effect on the fibrous composition. Cellulose degradation by some white rot fungi increased with cellulose content whereas cellulose degradation by other fungi was independent of cellulose content. All of the white rot fungi showed similar rates of lignin degradation, except for Pleurotus ostreatus, which was higher on M. sacchariflorus. The effect of the moisture contents of Miscanthus on cellulose and lignin decomposition by Phlebiopsis gigantea SPLog6 and Coniophora puteana 11E was also investigated. These results revealed subtle differences in the growth of white rot fungi on different Miscanthus genotypes.

Full Article

Biodegradation of Different Genotypes of Miscanthus by Wood Rot Fungi

Paul W. Baker,^a,* Ana Winters,^b and Mike D. C. Hale ^a

Miscanthus, which is comprised of several different genotypes, is an important high-biomass crop with applications in the biofuel industry and in the formation of biocomposite materials. The overall composition of Miscanthus can be altered via degradation with wood rot fungi. The starting composition revealed that the cellulose content of Miscanthus x giganteus was higher than that in Miscanthus sacchariflorus and that the lignin contents were similar in both genotypes. Of the wood rot fungi, only Lentinus edodes appeared to have completely colonized M. sacchariflorus and showed significant degradation. In contrast, all of the brown rot fungi showed partial colonization of both Miscanthus genotypes and had little effect on the fibrous composition. Cellulose degradation by some white rot fungi increased with cellulose content whereas cellulose degradation by other fungi was independent of cellulose content. All of the white rot fungi showed similar rates of lignin degradation, except for Pleurotus ostreatus, which was higher on M. sacchariflorus. The effect of the moisture contents of Miscanthus on cellulose and lignin decomposition by Phlebiopsis gigantea SPLog6 and Coniophora puteana 11E was also investigated. These results revealed subtle differences in the growth of white rot fungi on different Miscanthus genotypes.

Keywords: Miscanthus sacchariflorus; Miscanthus x giganteus; Lentinus edodes; Pleurotus ostreatus

Contact information: a: School of Natural Resources and Geography, Bangor University, Bangor, Gwynedd LL57 2UW, UK; b: Institute of Biological, Environmental, and Rural Sciences, Aberystwyth University, Reception, Penglais, Aberystwyth, Ceredigion, SY23 3FL, UK;

* Corresponding author: paul.baker@bangor.ac.uk

INTRODUCTION

Recently, there has been an interest in growing fibrous plants in the grassland areas of Wales for the production of bioethanol and biocomposite materials (Charlton et al. 2009). Miscanthus is one such high-biomass crop, whose fiber length is sufficient to form paper (Ververis et al. 2004), and which has been incorporated already as a component within mixed biocomposite boards (Park et al. 2012). The total lignin content of Miscanthus is generally much higher than in other agricultural crops, e.g., wheat straw, falling between 23.7% and 28.5%, which resembles the lignin content in soft and hard woods (Donaldson et al. 2001; Ververis et al. 2004; Goff et al. 2012). The majority of the lignin occurs in the outer ring of the M. x giganteus stem and decreases exponentially towards the pith (Kaack et al. 2003). In comparison to the stems, the leaves contain similar proportions of lignin but differ by having lower fiber content (Kim et al. 2012).

Most research has investigated the fungal biodeterioration of commercial wood, such as the discoloration caused by Trichoderma sp. (Huh et al. 2011) or the fungal degradation of timber (e.g. Nicolotti et al. 2010), but more recently the emphasis has shifted towards bioindustrial applications. The exposure of potential forestry waste to Coniophora puteana led to a decline in its total mass, resulting in an increased release of glucose (Ray et al. 2010), although scale-up with the prospect of biopulping using either the white rot fungus, Ceriporiopsis subvermispora, or the brown rot fungus, Postia placenta, showed little effect on lignin degradation (Giles et al. 2012). Another study identified Trametes velutina in being effective in delignifying Populus tomentosa after 12 weeks incubation (Wang et al. 2014). In addition to wood, white rot fungi grows on a wide range of lignocellulose materials originating from agricultural waste that would otherwise present problems in disposal, and by means of the fungi these materials can be turned into useful products such as biofuels, animal feeds, or paper, or further biorefined into potentially valuable products (Sánchez 2009; Menon and Rao 2012). The growth of different types of white rot fungi has been examined on corn stover (Shrestha et al. 2008), wheat straw (Hatakka 1983; Moyson and Verachtert 1991; Wan and Li 2010; Arora et al. 2011; Shrivastava et al. 2011), rice straw (El-Din et al. 2013), ula grass (Carex meyeriana) (Mao et al. 2013), and Bermuda grass (Gamble et al. 1994).

Few studies have examined the growth of wood rot fungi on Miscanthus. In Osono’s study, Trametes versicolor exhibited the most active degradation of leaves, at 43% of the total mass, when the leaves were laid onto 2% agar for 12 weeks. However, the leaves contain easily biodegradable components (Kim et al. 2012) that are perhaps readily accessible, whereas the composition of Miscanthus stems is quite different. It was calculated that T. versicolor had degraded 46% of the lignin, but the amount of lignin remaining in the degraded material showed little change from the original starting material. In another study, fungi associated with M. x giganteus were isolated, and studies of its degradation showed that some Ascomycota were principally involved, although it was concluded that they were unlikely to be as effective as white rot fungi under suitable conditions (Shrestha et al. 2011). A recent study using three white rot fungi revealed that growth occurred causing changes in the fibrous composition that varied depending on the fungus (Baker et al. 2015).

The aim of this study was to examine the biodegradation of M. sacchariflorus and M. x giganteusby different wood rot fungi. The effects would be assessed based on dry weight loss, degradation of cellulose, and degradation of lignin. The fungi used in the study were comprised of six white rot fungi, three brown rot fungi, and a fungus involved typically in the discoloration of wood, Trichoderma sp.

EXPERIMENTAL

Preparation of Microcosms

Three types of Miscanthus genotypes – M. sacchariflorus with low fibrous content (LF); M. giganteus x M. sacchariflorus x M. sinensis, sterile triploid with high fibrous content (HF); and M. sacchariflorus x M. sinensis, tetraploid were provided by the Institute of Biological, Environmental, and Rural Sciences (IBERS), Aberystwyth University. The plants were established from rhizome material in late spring of 2005 and planted in replicate plots of 25 m²with 49 plants per plot. The plant material was harvested in March 2011 and roughly chipped, air-dried, and stored in a dry environment. The material (10% w/v) was thoroughly mixed in hot distilled water at 80 °C, left for 30 min, and sieved. Then, 20 g of wet plant material containing 3.851 ± 0.008 g of oven-dry weight Miscanthus was placed into each glass jar and sealed with a metal lid containing a 10-mm hole that was plugged with cotton wool to allow for gaseous exchange. The glass jars were autoclaved for 60 min at 121 °C and 103.4 kPa. When the glass jars had cooled, they were inoculated in triplicate with agar squares excised from fungal cultures that had grown for one week on malt extract agar plates. Three jars remained un-inoculated to act as the control. All fungi used in this study were kept at the Bangor University culture collection. The fungi used in this study were white rot fungi (Lentinus edodes CYN, Pleurotus ostreatus Pox K, Phlebiopsis gigantea SPLog6 (Forest Research), Ganoderma australe GA1, and Ganoderma lucidum GL1), brown rot fungi (Fibroporia vaillantii (FPRL14A), Coniophora puteana 11E, and Gloeophyllum trabeum (MUCL 11353; BAM 109; FPRL 108N; MUCL 11663), and an Ascomycete (Trichoderma CP021). Each of the glass jars was weighed empty and then weighed again when it was filled with the autoclaved Miscanthus and the inoculant. The glass jars were incubated in the dark, in an environmentally controlled room at 22 °C, and at 65% humidity for four weeks. On the final day, the glass jars were weighed, and the samples were removed to determine their moisture content. The remainder was placed into a blender for 1 to 2 min until fine particles were apparent, then oven-dried at 105 °C for 24 h and used for subsequent analyses.

To determine the effects of moisture content on the growth of fungi, another experiment was performed in which increasing amounts of water were added to the different microcosms. A sample of air-dried M. sacchariflorus x M. sinensis (5.22 ± 0.013 g) was placed into each glass jar, and 5.75 (omitted for C. puteana 11E), 10.45, 15.17, 19.91, 24.59, and 29.39 g of distilled water was added to each in triplicate. These microcosms were autoclaved and inoculated as previously described, except that they were either inoculated with either P. gigantea SPLog6 or C. puteana 11E. A yeast extract (1% w/w) was included in the microcosms that were inoculated with C. puteana 11E in order to encourage mycelial growth throughout the jars. The microcosms were incubated and destructively analyzed as previously described.

Analysis of Microcosms and Original Plant Material

The wet weight of the Miscanthus in the microcosms was measured, and samples were collected for a determination of the moisture content before autoclaving. The moisture lost during autoclaving was taken into account to accurately calculate the moisture content of the microcosms at the start of the experiment. At the end of the experiment, a sample was removed from each microcosm in the region where the fungus had colonized the Miscanthus. The sample was oven-dried at 105 °C for 24 h to determine the moisture content, as previously described for wood (Knight 1931). Once the water content was calculated, it became possible to determine the dry mass remaining in each microcosm after weighing the total wet mass. The fiber content was determined by means of acid detergent fiber extraction using an Ankom 2000 fiber analyzer (Ankom Technology, New York, USA). Briefly, 0.5 g of oven-dried Miscanthus was placed into each filter bag that was then heat-sealed, and a filter bag without Miscanthus was included as a blank control. The filter bags were washed in a solution of 2 g/L cetyl trimethylammonium bromide and 1 N sulphuric acid (Lowry et al. 1994) and processed as described in the manufacturer’s instructions (Ankom). Lignin was extracted from a 0.3 g oven-dried Miscanthususing the Klason method (Hatfield et al. 1994) with a minor modification, as 3 h of incubation at 20 °C with 5 mL of 72% (v/v) sulphuric acid was found to result in higher digestion than 2 h of incubation. The ash content was determined by heating 1 g of dry Miscanthus (undegraded and degraded by each of the fungi) in a muffle furnace for 4 h. The cellulose percentages were calculated by subtracting the lignin and ash percentages from the fiber percentages. The percentages of lignin were calculated by subtracting the ash percentages. Finally, the percentages of cellulose and lignin were obtained by normalizing to the microcosms that remained uninoculated and showed no changes in fibrous content.

Statistics

Using IBM SPSS Statistics version 20, statistical analyses in the form of ANOVA followed by Tukey’s post hoc test were performed on the values of composition of each Miscanthus genotype and on data obtained using different moisture contents. Statistical analysis of the mass loss percentages was determined using t-Test for each fungus degrading both Miscanthus genotypes.

RESULTS AND DISCUSSION

Composition of Miscanthus Genotypes before Degradation

The cellulose content of M. x giganteus was significantly different from the cellulose contents of the other two genotypes, M. sacchariflorus and M. sacchariflorus x M. sinensis (Fig. 1). Lignin extracted from both Miscanthus genotypes showed that there were no significant differences between them. These results correlated well with those in previous studies (Hodgson et al. 2010). The percentages of lignin recovered were perhaps a little higher than those previously described using the Klason method (Ververis et al. 2004; Goff et al. 2012). The temporary storage of M. giganteus may have marginally increased the percentages of cellulose and lignin caused by the losses in volatiles or by degradation by Ascomycetes (Graham et al. 2012). However, once the Miscanthus was harvested, dried, and chipped, it was used within a few months, so it was unlikely that Ascomycetes caused much degradation.

Fig. 1. The percentages of cellulose and direct Klason lignin extraction were determined in the autoclaved M. sacchariflorus LF (M. sac), the M. sacchariflorus x M. sinensis (M. sac x M. sin), and the Miscanthus x giganteus (M. x gig).

Effects of Fungi on Dry Mass

The fungal degradation of M. sacchariflorus and M. x giganteus in terms of mass loss from each microcosm can be found in the Appendix (Table 1). L. edodes CYN, P. ostreatus Pox K, P. gigantea SPLog6, G. lucidum GL1, and Trichoderma CP021 formed visible mycelia that clearly colonized the entire jar containing both genotypes of Miscanthus, whereas G. australe GA1, F. vaillantii FPRL 14A, C. puteana 11E, and G. trabeum FPRL 108N only colonized Miscanthusaround the point of inoculation. Some fungi, such as L. edodes CYN in M. sacchariflorus and C. puteana 11E in M. x giganteus, appeared to show greater degradation of the plant material compared to other fungi (Fig. 2).

Statistical analyses revealed that the dry weight associated with L. edodes CYN was significantly lower than that of G. trabeum 108N, and that the dry weight associated with C. puteana 11E was significantly lower than that associated with G. australe GA1 and F. vaillantii 14A. The dry weight losses showed large errors, and it was speculated that due to the heterogeneity of the grass material, the small portion sampled might have only crudely reflected the total amount degraded in each microcosm. Consequently, smaller weight losses that might have occurred would not have been detected, and a previous study with P. gigantea grown on wheat straw revealed a low weight loss (Salvachúa et al. 2011).

Effect of Fungi on Cellulose

Assuming there is an inverse correlation between cellulose and hemicellulose in the various Miscanthus genotypes, it would be expected that these hemicelluloses would be degraded more quickly in M. x giganteus than in M. sacchariflorus. None of the cellulose data showed any significant differences between the different fungi (Fig. 2 and Table 1). When the percentages of cellulose degradation were calculated, P. gigantea SPLog6, G. australe GA1, and G. lucidumGL1 showed higher degrees of cellulose degradation on M. x giganteus than on M. sacchariflorus, and this was found to be significant using the t-test (Fig. 2). Therefore, cellulose degradation appeared to be directly correlated with the cellulose content of each genotype of Miscanthus.

These results obtained with P. gigantea compared well with the results of another strain grown on wheat straw that resulted in high cellulose degradation (Salvachúa et al. 2011). P. gigantea is considered effective at degrading all components of lignocellulosic materials (Hori et al. 2014), whereas Ceriporiopsis subvermispora is considered a more selective degrader of lignin (Wan and Li 2010). However, especially large differences in the proportion of cellulose that was degraded were observed between G. australe GA1 and G. lucidum GL1, perhaps reflecting high cellulase activities occurring in M. x giganteus in response to the higher cellulose contents. It would appear that these fungi are not selective lignin degraders. However, not all fungi showed the same response to the higher content of cellulose in Miscanthus. In contrast, the extent of cellulose degradation by L. edodes CYN was similar in both genotypes, which would seem to indicate that the presence of more cellulose in M. x giganteus did not dramatically increase the cellulase activity. Consequently, it would appear that as the cellulose content increased within Miscanthus, the fungi such as L. edodes CYN that exhibited lower levels of cellulase activity may have produced a degraded product that had lower lignin content than that of other fungi.

Therefore, it would appear that among the fungi used in this study, L. edodes CYN showed the most selective lignin degradation when grown on Miscanthus. Its growth on other lignocellulosic materials may have resulted in the expression of other isoenzymes involved in cellulose degradation.

Fig. 2. Changes in the percentages of dry weight cellulose, and lignin from the original proportions present in the M. sacchariflorus LF genotype (yellow bars) and M. x giganteus HF genotype (orange bars) after degradation by different fungi: (LE) Lentinus edodes CYN, (PO) Pleurotus ostreatus Pox K, (PG) Phlebiopsis gigantea SPLog6, (GA) Ganoderma australe GA1, (GL) Ganoderma lucidum GL1, (FV) Fibroporia vaillantii 14A, (CP) Coniophora puteana 11E, (GT) Gleophyllum trabeum FRRL 108N, and (T) Trichoderma sp.

Effects of Fungi on Lignin

An analysis of the lignin contents remaining in the fungi-degraded material revealed that the fungal degradation of the M. sacchariflorus LF genotype could be divided into two distinct groups (Fig. 2 and Table 1). Significantly higher proportions of lignin were degraded in the microcosms inoculated with L. edodes CYN, P. ostreatus Pox K, P. gigantea, and G. lucidum GL1 compared with the microcosms inoculated with G. australe GA1, F. vaillantii 14A, C. puteana11E, G. trabeum 108N, and Trichoderma sp. CP021. The fungal degradation of the M. x giganteus HF genotype showed that L. edodes CYN, P. ostreatus Pox K, P. gigantea SPLog6, G. australe GA1, G. lucidum GL1, and Trichoderma sp. CP021 all achieved higher percentages of lignin degradation than did C. puteana 11E. However, L. edodes CYN and P. gigantea also showed higher percentages of lignin degradation compared to P. ostreatus Pox K, G. australeGA1, F. vaillantii 14A, C. puteana 11E, G. trabeum 108N, and the uninoculated control. The percentages of lignin degradation achieved by P. ostreatus Pox K and F. vaillantii 14A grown on M. sacchariflorus were significantly higher than those grown on M. x giganteus. In contrast, L. edodes CYN, P. gigantea SPLog6, and G. lucidum GL1 showed no significant differences in lignin degradation when grown on M. sacchariflorus versus M. x giganteus.

In contrast to the indigestible lignin that is obtained in other studies when lignin is extracted after the recovery of fiber, in this study lignin was extracted directly from Miscanthus. The same direct procedure was used to recover lignin from Miscanthus leaves that had been degraded by white rot fungi, and although T. versicolor had degraded a significant proportion of lignin, the proportion of lignin that remained appeared to show little change (Osono 2010). Other, less-studied fungi, such as Marasmius sp. and Coccomyces sp., appeared to be far more effective in removing up to 38% of the lignin from the remaining partially degraded Miscanthus leaves. One possible reason for this was the ready availability of nutrients in the Miscanthus leaves, in contrast to the nutrients present in chopped Miscanthus stems, which are inaccessible until delignification occurs. Previous studies have suggested that up to 30% of the indigestible lignin in wheat straw can be degraded by different white rot fungi (Singh et al. 2011; Knežević et al. 2013), and it would appear that considerably less delignification occurs in Miscanthus. In these studies, a variety of modifications were used that involved either inoculation using spores or the use of a mineral medium that could promote fungal colonization. It was found that the protein content in another genotype of Miscanthus was about 2.4% (Huyen et al. 2010), which is quite high compared to the protein content of other agricultural materials, e.g., corn stover and hardwoods (Duncan and Schilling 2010). High nitrogen contents are known to have a negative effect on lignin-degrading activity (Reid 1983). It may be possible to increase delignification by incubating fungi such as L. edodes CYN and C. subvermispora, which appear to show more selective lignin degradation, on Miscanthus for longer periods.

Effects of Moisture Content on Fungal Growth

The mass of the Miscanthus degraded by P. gigantea SPLog6 appeared to increase with increases in moisture content. However, statistical analyses revealed that although there were no significant differences between the smaller microcosms (6 g dry weight), in the larger microcosms (30 g dry weight), significantly higher decomposition occurred among those with moisture contents of 227% and 406% compared with the microcosms with moisture contents of 43%. Cellulose degradation was significantly higher in the microcosms with moisture contents of 78% compared with those with moisture contents of 309% and 406%.

Fig. 3. The effect of different moisture contents on the percentages of dry-weight cellulose and lignin remaining in M. sacchariflorus x M. sinensis degraded by (PG) (P. gigantea SPLog6) and CP (C. puteana 11E). (PG2) are microcosms containing 100 g wet Miscanthus; (PG) and (CP) contain 30 g Miscanthus.

In the microcosms that had moisture contents of 227%, 309%, and 390%, respectively, the amount of lignin degraded was 8.3 ± 1.1%, 6.7 ± 0.5%, and 6.8 ± 1.9%. Significantly more lignin was degraded in these microcosms than in the microcosms with moisture contents of 78%. Although some white rot fungi can affect degradation using radical generation, biomass is generally degraded using a combination of lignin-modifying peroxidases (manganese peroxidase, lignin peroxidase, and versatile peroxidase), laccases, oxalic acid, and hydrogen peroxide (Lundell et al. 2010), and lower moisture contents would ensure that enzymes were in close proximity to the substrates. To retain similar levels of enzyme activity, higher concentrations of enzymes may need to be produced when moisture conditions increase.

C. puteana 11E appeared to show increases in decomposition with increases in moisture content, although statistical analyses revealed no significant differences. Although more cellulose appeared to be degraded in the microcosms with moisture contents of 475%, neither cellulose nor lignin showed any significant differences in degradation within microcosms of any particular moisture content compared to others. However, statistical analyses of the fiber contents in the microcosms with moisture contents of 475% showed that significantly more fiber had been degraded than in microcosms with moisture contents of 154%, 309%, and 390% (data not shown). Brown rot fungi uses oxidative systems to degrade biomass during the initial stages of decomposition (Baldrian and Valášková 2008), which may show optimal activity when the biomass particles are in close proximity to each other. In fact, milled corn stover has been the only non-wood material where the growth of brown rot fungi was investigated (Duncan and Schilling 2010).

CONCLUSIONS

1. There were clear differences between white rot fungi which completely colonized M. sacchariflorus causing changes in the fibrous composition, whereas brown rot fungi (Fibroporia vaillantii 14A, Coniophora puteana 11E, Gleophyllum trabeum FRRL 108N) only partially colonized M. sacchariflorus. Among the white rot fungi, L. edodes CYN was found to cause the most degradation of M. sacchariflorus.
2. P. gigantea SPLog6 and both Ganoderma sp. (G. australe GA1 and G. lucidum GL1) appeared to degrade more cellulose as the quantity of cellulose present in the Miscanthusgenotypes increased. In contrast, the other fungi analyzed in this study (L. edodes CYN and P. ostreatus Pox K) showed no significant differences in the extent of cellulose degradation between the two different Miscanthus genotypes.
3. P. ostreatus Pox K showed higher delignification when grown on the M. sacchariflorus than on M. giganteus, which was accounted for by the lower cellulose content. Other white rot fungi did not differ in the extent of delignification between the two Miscanthusgenotypes.
4. Generally white rot fungi showed low levels of delignification in Miscanthus compared with other lignocellulosic substrates, although small improvements could be made such as moisture content optimization. Other physiological parameters may have further impacts on delignification and conditions could be strictly controlled in future studies using a solid state bioreactor.

ACKNOWLEDGMENTS

The research was carried out within a BEACON project funded by the European Regional Development Fund through the Welsh Government.

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APPENDIX

Table 1. Percentages of fiber, cellulose, and lignin remaining in the M. sacchariflorus LF genotype (LF) and M. x giganteus HF genotype (LF) after degradation by different fungi*

* (LE) Lentinus edodes CYN, (PO) Pleurotus ostreatus Pox K, (PG) Phlebiopsis giganteaSPLog6, (GA) Ganoderma australe GA1, (GL) Ganoderma lucidum GL1, (FV) Fibroporia vaillantii 14A, (CP) Coniophora puteana 11E, (GT) Gleophyllum trabeum FRRL 108N, and (T) Trichoderma sp. The columns showing t-test are statistical analysis between LF and HF samples for each fungus and those in bold are significantly different.

Article submitted: July 9, 2015; Peer review completed: September 6, 2015; Revised version received: October 7, 2015; Accepted: March 18, 2016; Published: March 30, 2016.

DOI: 10.15376/biores.11.2.4379-4391