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Temporal changes in wood crystalline cellulose during degradation by brown rot fungi
International Biodeterioration & Biodegradation 63 (2009) 414–419
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Temporal changes in wood crystalline cellulose during degradation by brown rot fungi Caitlin Howell a, *, Anne Christine Steenkjær Hastrup b, Barry Goodell c, Jody Jellison a a
School of Biology and Ecology, University of Maine, 311 Hitchner Hall, Orono, ME 04469, USA Department of Microbiology, University of Copenhagen, Sølvgade 83H, 1307 Copenhagen K, Denmark c Department of Wood Science and Technology, 5755 Nutting Hall, Orono, ME 04469, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2008 Received in revised form 24 November 2008 Accepted 26 November 2008 Available online 11 January 2009
The degradation of wood by brown rot fungi has been studied intensely for many years in order to facilitate the preservation of in-service wood. In this work we used X-ray diffraction to examine changes in wood cellulose crystallinity caused by the brown rot fungi Gloeophyllum trabeum, Coniophora puteana, and two isolates of Serpula lacrymans. All fungi increased apparent percent crystallinity early in the decay process while decreasing total amounts of both crystalline and amorphous material. Data also showed an apparent decrease of approximately 0.05 Å in the average spacing of the crystal planes in all degraded samples after roughly 20% weight loss, as well as a decrease in the average observed relative peak width at 2q ¼ 22.2 . These results may indicate a disruption of the outer most semi-crystalline cellulose chains comprising the wood microfibril. X-ray diffraction analysis of wood subjected to biological attack by fungi may provide insight into degradative processes and wood cellulose structure. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: X-ray diffraction Crystallinity Serpula lacrymans Coniophora puteana Gloeophyllum trabeum Wood decay
1. Introduction Wood cell walls consist of cellulose, hemicelluloses, lignin, pectin, proteins, and other trace materials. Cellulose, which comprises 46–52% of the wood volume in softwoods and 39–52% in hardwoods (Zabel and Morrell, 1992), is composed of linear chains of b-1,4 linked D-anhydroglucopyranose, with cellobiose as the repeating unit (O’Sullivan, 1997). These chains are arranged into elementary fibrils, consisting of 60–70% crystalline cellulose and 30–40% amorphous cellulose, and surrounded by a hemicellulose and lignin matrix (Salmen and Olsson, 1998; Neagu et al., 2006). The crystalline portions of wood are formed when the cellulose chains that comprise the microfibril are close enough to form hydrogen bonds, creating a regularly patterned structure (O’Sullivan, 1997). Recent atomic force microscopy (AFM) work by Ding and Himmel (2006) on corn stover has resulted in a refined model of the arrangement of cellulose chains in the elementary fibril. In this model, the six innermost cellulose chains are the most highly ordered and display an Ib cellulose structure. These chains are surrounded by 12 slightly less crystalline (sub-crystalline) chains,
* Corresponding author. Tel.: þ1 207 581 3032; fax: þ1 207 581 2537. E-mail address: [email protected] (C. Howell). 0964-8305/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2008.11.009
which are surrounded in turn by 18 less ordered (semi-crystalline or non-crystalline) cellulose chains. In wood, hemicelluloses are thought to have a closer association with cellulose than lignin (Salmen, 2004; Lawoko et al., 2005; Neagu et al., 2006). Nevertheless, the exact structure of the cellulose microfibrils, macrofibrils, and the associations of these with hemicelluloses and lignin remain unclear. X-ray diffraction (XRD) has been used for decades as a rapid, non-destructive method for observing the crystalline portion of wood (Cullity, 1978; Cave, 1997; Lichtenegger et al., 1999). The interference pattern created by the wood can be used to determine the overall percent of the wood that is in a crystalline state by comparing the sharp crystalline diffraction signals to the broad signals created by the amorphous material. A number of studies have focused on the modification of crystalline cellulose by brown rot wood decay fungi as characterized by XRD. Those focusing on the depolymerization of cellulose by the brown rot fungus Postia placenta have shown that early in the decay process this fungus will temporarily increase overall crystallinity and the size of the crystallites, presumably due to a preferential degradation of the amorphous regions of the cellulose microfibrils (Highley et al., 1988; Klemen-Leyer et al., 1992). The purpose of this study was to thoroughly examine changes in wood crystallinity caused by three different species of brown rot
C. Howell et al. / International Biodeterioration & Biodegradation 63 (2009) 414–419
fungi over time, and to compare the resulting data to changes observed in weight loss and wood sugar composition. 2. Materials and methods 2.1. Growth conditions Fungi were maintained at 21 C on 2% (w/v) potato dextrose agar (PDA) until inoculation. Fungi used in this study were Serpula lacrymans (Wulfen: Fries) Schro¨ter (1889) Bb 29 from the Technological Institute, Denmark, and Sl 221 provided by Dr H. Kauserud, Norway, Coniophora puteana (Schumach.) P. Karst. (1868) ATCC 44393 and Gloeophyllum trabeum (Pers.:Fr.) Murrill (1908) ATCC 11539. C. puteana and G. trabeum cultures were purchased from the American Type Culture Collection (Manassas, VA, USA). 2.2. Soil block assays Four 1-cm2 pieces of inoculum were taken from the outer edge of mycelium of 3-week-old fungal cultures and placed into modified AWPA soil block jars (AWPA, 2003). Each piece of inoculum was placed at one corner of a pair of birch feeder strips on top of approximately one cup of a 1:1:1 mixture of potting soil, sphagnum peat moss, and horticultural grade vermiculite, hydrated with deionized water. One 1.2 2.5 2.5 cm transverse pine block was placed in the jar after the mycelial mat had covered the set of birch feeder strips. Fungi were allowed to grow for 1, 2, 4, 6, 8, 10, and 12 weeks after the addition of the wood block. There were five replicates per fungus per harvest, as well as five uninoculated controls.
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least-squares peak fitting method with an amorphous standard (O’Sullivan, 1997; Andersson et al., 2003; Thygesen et al., 2005), a Rietveld analysis (Rietveld, 1967, 1969) using the cellulose 1b crystal structure published by Nishiyama et al. (2002), and an area comparison method. No significant differences were found for percent crystallinity calculated by the three methods. Percent crystallinity was calculated from raw XRD spectra by comparing the area of the crystalline regions to the total area. Removal of crystalline material over time was calculated by multiplying the calculated percent crystallinity of a block by the measured dry weight of the block. Removal of amorphous material over time was calculated by multiplying the percent amorphous material (defined as 100%dpercent crystallinity) by the total dry weight of the block. Using this method it was possible to adjust for percent weight loss in calculating the decrease in crystalline and amorphous material over time. Ratio calculations such as percent crystallinity values were not adjusted. Adjusting these values for weight loss would have caused the loss of information and yielded an inaccurate representation of percent crystallinity. 2.5. Wood sugar and Klason lignin analysis Carbohydrate analysis of the ground material of each replicate within a sampling time set was analyzed using high pH anionexchange chromatography with pulsed amperometric detection (HPAEC/PAD) according to Davis (1998). The amount of acidinsoluble lignin in the wood material was analyzed according to the American Wood Preservers’ Association (AWPA) method D1106-96 (AWPA, 1996). 2.6. Statistics
2.3. Processing and X-ray diffraction All blocks were dried for 48 h at 95 C and weighed immediately after harvest. Blocks were ground in a Wiley Mill to pass through a standard 40-mesh screen (420 mm) and pressed into a cylindrical wafer 25 mm in diameter and 4.3-mm thick. Wood wafers were scanned using a Panalytical X’Pert X-Ray Diffraction machine (Panalytical, Netherlands) with symmetric q–2q Bragg–Brentano scattering geometry. Nickel filtered Ka radiation with a wavelength of 1.542 Å was used to perform q–2q scans. The incident beam was passed through a fixed divergence slit of 1/16 and an anti-scatter slit of 1/8 , as well as a 15-mm mask. The diffracted beam was passed through a size 5.0 anti-scatter slit. Data were collected in the 2q-range 5–35 with a step size of 0.01. The scans proceeded at 0.08 per second (150 s per step). 2.4. XRD spectral analysis Due to the variability of published procedures on the analysis of XRD spectra from wood, the spectra from these experiments were processed and analyzed using three different methods: a standard
Statistical analyses of weight losses, percent crystallinity, crystallite size, simple sugar concentrations and d-spacing values were performed employing either one-way or two-way ANOVAs and protected Fisher LSD post hoc tests using SySTAT v.12 (Systat Software Inc., San Jose, CA, USA). 3. Results and discussion 3.1. Weight loss, HPLC and Klason lignin analysis Blocks decayed by all four strains of brown rot fungi showed significant weight losses after 12 weeks. G. trabeum degraded the pine blocks most quickly, reaching a value of 17.2% weight loss after only 2 weeks of decay. Weight losses in blocks decayed by the other fungi tested reached approximately 20% weight loss after 4 weeks (Table 1). Quantification of wood sugars and Klason lignin yielded results consistent with previously published data on wood carbohydrate removal by brown rot fungi, which show hemicellulose depletion up to approximately 20% of weight loss, followed by cellulose
Table 1 Weight loss values for wood blocks inoculated with S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt). The control represents uninoculated wood blocks. Values in parenthesis indicate standard deviations. The asterisks (*) denote unavailable data. Fungal species
Week 1
2
4
6
8
S. lacrymans (Bb) S. lacrymans (Sl) C. puteana (Cp) G. trabeum (Gt) Control (K)
0.7 (1.2) 0.3 (0.1) 0.1 (0.5) 1.8 (0.3) 0.2 (0.4)
2.8 (0.0) 4.1 (0.9) 3.2 (0.9) 17.2 (2.4) 0.0 (0.1)
16.8 (4.8) 21.7 (4.0) 18.4 (2.2) 41.2 (6.2) 0.0 (0.1)
26.3 (5.5) 33.1 (6.7) 25.3 (4.9) 57.7 (1.0) 0.0 (0.0)
32.8 49.4 25.2 59.5 0.0
(7.8) (2.0) (6.8) (2.3) (0.1)
10
12
* 53.8 (3.5) 28.7 (5.5) 56.9 (4.2) *
49.9 (4.5) 58.5 (2.4) 39.5 (11.7) 61.3 (6.1) 0.2 (0.2)
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% of remaining material
S. lacrymans Bb
S. lacrymans Sl
45 40 35 30 25 20 15 10 5 0
wk 0 wk 2 wk 4 wk 6
Glucan
Xylan
45 40 35 30 25 20 15 10 5 0
wk 0 wk 2 wk 4 wk 6
Mannan
Glucan
% of remaining material
G. trabeum wk 0 wk 2 wk 4 wk 6
Xylan
Mannan
C. puteana
45 40 35 30 25 20 15 10 5 0 Glucan
Xylan
45 40 35 30 25 20 15 10 5 0
wk 0 wk 2 wk 4 wk 6
Mannan
Glucan
Xylan
Mannan
Fig. 1. Glucan, xylan and mannan wood sugar analysis data for wood blocks inoculated with S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393, and G. trabeum ATCC 11539 after 0, 2, 4 and 6 weeks.
depletion for the remaining weight loss (Curling et al., 2001). The proportional removal of both hemicellulose and cellulose sugars was consistent for all fungi tested with the exception of C. puteana, which appeared to remove more xylan and mannan and less glucan at comparable weight loss values (Fig. 1). Analysis of Klason lignin levels showed an increasing percent of lignin in the remaining material as the weeks progressed (Table 2), consistent with the known degradation methods of these fungi. 3.2. Percent crystallinity and removal of crystalline and amorphous material over time All fungi showed significant increases in percent crystallinity, or percent of the residual material that was in a crystalline state, compared to the controls as early as week 1 (P < 0.022) (Fig. 2). Percent crystallinity values for the two isolates of S. lacrymans and C. puteana were significantly different by week 4 of decay (P ¼ 0.000), while values for G. trabeum had begun to decline by this point. By week 12, G. trabeum and both S. lacrymans isolates yielded percent crystallinity values lower than the controls, although only G. trabeum was significantly different (P ¼ 0.001). This trend (an increase in crystallinity early in the decay process, followed by a decrease) has been previously observed by other researchers examining wood decayed by fungi (Highley et al., 1988; Klemen-Leyer et al., 1992) and wood treated chemically to extract hemicelluloses (Hult et al., 2003; Åkerholm et al., 2004), and has been attributed to the initial removal of hemicelluloses and other amorphous materials. The observed increase in crystallinity peaks
around week 2 for wood degraded by G. trabeum, and around week 4 for the other fungi as weight losses increase to approximately 20%, a level consistent with the near complete removal of the hemicelluloses (Curling et al., 2001). After week 2 (for G. trabeum) and week 4 (for all other isolates), the percent crystallinity values begin to decrease, possibly due to continuing fungal attack on the crystalline cellulose only after degradation of the more readily available amorphous nutrient sources. Percent crystallinity values for G. trabeum dip below the values for the controls by week 12. This is possibly due to the fact that amorphous lignin is being left behind and comprises a greater percentage of the remaining material as the weeks progress (Table 2). The percent crystallinity values for C. puteana remained above the level of the controls at week 12. This may be due to the relatively low weight loss values achieved by this fungus (39.5%) as compared to the other fungi at this time point. Calculation of the rates of removal of amorphous versus crystalline material revealed that the degradation of the amorphous material began immediately, as confirmed by the HPLC data (Fig. 1). Total amounts of crystalline material began to decrease at week 2 for G. trabeum and week 4 for the other fungi (Fig. 3), again as the samples approached the 20% weight loss benchmark which signals nearly complete degradation of the easily accessible hemicelluloses. The amount of amorphous material appeared to remain constant after week 6 for G. trabeum and after week 8 for S. lacrymans isolate Sl 221, while the relative amount of crystalline material continues to decrease after this point. This may be due to the achievement of a steady state in which the crystalline material is
Table 2 Klason lignin (give in percent of remaining material with standard deviations in parentheses) for wood blocks inoculated with S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt). The control represents uninoculated wood blocks. The asterisks (*) denote unavailable data. Fungal species
S. lacrymans (Bb) S. lacrymans (Sl) C. puteana (Cp) G. trabeum (Gt) Control (K)
Week 1
2
30.5 (0.3) 30.7 (0.4) 30.6 (0.6) 30.1 (1.2) 30.6 (2.1)
30.0 30.8 28.6 33.8 30.0
4 (1.8) (0.7) (0.5) (1.0) (1.4)
35.0 36.7 36.0 48.8 28.8
(1.2) (2.4) (1.0) (3.7) (0.2)
6
8
10
12
39.1 (2.3) 42.7 (3.1) 38.2 (2.5) 61.6 (2.6) 28.5 (0.4)
* 54.4 (2.3) 39.6 (2.3) * *
* 61.0 (3.8) 40.9 (1.6) 64.2 (7.8) 29.1 (0.8)
59.1 (3.1) 66.6 (2.3) 44.0 (6.8) 68.9 (8.7) 28.4 (0.5)
C. Howell et al. / International Biodeterioration & Biodegradation 63 (2009) 414–419
% Crystallinity
S. lacrymans Bb 55 50 45 40 35 30 25 20 15
K Bb
S. lacrymans Sl
55 50 45 40 35 30 25 20 15
K Sl Week Week Week Week Week Week Week Week 0 1 2 4 6 8 10 12
Week Week Week Week Week Week Week Week 0 1 2 4 6 8 10 12
C. puteana
% Crystallinity
G. trabeum 55 50 45 40 35 30 25 20 15
55 50 45 40 35 30 25 20 15
K Gt
417
K Cp Week Week Week Week Week Week Week Week 0 1 2 4 6 8 10 12
Week Week Week Week Week Week Week Week 0 1 2 4 6 8 10 12
Fig. 2. Percent crystallinity in uninoculated control wood blocks (K, dashed lines), and wood blocks inoculated with S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt) (solid lines).
converted into amorphous material before absorption and ultimate metabolism by the fungi. This conversion seems to occur at or near weight loss levels of 50%. These trends are not observed in wood degraded by S. lacrymans isolate Bb 29 and C. puteana, as these fungi either did not achieve the appropriate weight loss levels early enough in the study period, or at all. 3.3. Average d-spacing Analysis of the peak locations showed a small but consistent and significant rightward shift in the 200 peak in XRD spectra of the wood blocks decayed by fungi, indicating a decrease in the average d-spacing of these crystalline planes. Calculations revealed that the average spacing between the crystalline planes was reduced significantly at week 1 for G. trabeum and C. puteana (P ¼ 0.047 and P ¼ 0.046, respectively) and by week 2 for the two isolates of S.
lacrymans (P ¼ 0.031 (Sl) and P ¼ 0.002 (Bb)). Although the overall change in d-spacing was minimal (approximately 0.05 Å), it was found to be similar for all fungi. This decrease became significant (P ¼ 0.000) at the 20% weight loss plateau in all cases and remained at that level through week 12 of decay (Fig. 4). Slight shifts in the location of the 200 peak in cellulosic material have previously been observed by other researchers, most notably between cellulose from sources classified as Ia-dominant, and those classified as Ib-dominant (Wada et al., 2001). The International Center for Diffraction Data (ICDD) lists the cellulose 200 peak as having a maximum intensity at 21.80 2q for Ia, and at 22.98 for Ib (ICDD, 2008). It is possible, therefore, that the shifts observed in these experiments and attributed to fungal degradation indicate a move toward a more Ib-dominant system. This would be consistent with the model developed by Ding and Himmel (2006) who observed that the more highly ordered chains at the center of
Control Amorphous Decayed Amorphous
Material (g)
2.5
S. lacrymans Bb
Control Crystalline Decayed Crystalline 2.5
2
2
1.5
1.5
1
1
0.5
0.5 0
0 wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
Material (g)
2.5
S. lacrymans Sl
G. trabeum
wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12 2.5
2
2
1.5
1.5
1
1
0.5
0.5
C. puteana
0
0 wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
Fig. 3. Amount of crystalline material (light colors) versus amorphous material (dark colors) in wood blocks degraded by S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt). Grey dotted lines represent values calculated for control blocks harvested at the same time as the inoculated blocks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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C. Howell et al. / International Biodeterioration & Biodegradation 63 (2009) 414–419
S. lacrymans Bb d-spacing ( Å)
8.15
S. lacrymans Sl 8.15
8.1
8.1
8.05
8.05
8
8
7.95
7.95
7.9
K
7.85
wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
G. trabeum
8.15
d-spacing ( Å)
Sl
7.85 wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
C. puteana 8.15
8.1
8.1
8.05
8.05 8
8 7.95
K
7.9
Bb
K
7.95
Gt
7.9
K Cp
7.85
7.9 wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
Fig. 4. Average d-spacing values between the cellulose crystal planes in uninoculated control wood blocks (K), and wood blocks decayed by S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt).
the cellulose elementary fibrils display an Ib conformation. The fact that this shift occurs early in the decay process and is constant throughout the remaining weeks may be another indication of the fluidity and availability of the surface chains and, conversely, the rigidity of the more crystalline inner chains. It is interesting to note that significant changes in percent crystallinity and d-spacing, as measured by XRD, are detectable relatively early in the decay process for all fungi tested. Previous studies using wood degraded by G. trabeum have shown that significant decreases in strength properties occur even before significant weight loss is detected (Curling et al., 2001). It has been established that this is most likely due to extensive cellulose and hemicellulose depolymerization (Goodell, 2003). Although this change is most likely to be detectable in the lengthwise direction of the microfibril, it is also possible that the changes in FWHM and 2q values seen in these experiments are a result of the depolymerization process. Also noteworthy is the similarity of the percent crystallinity and weight loss values documented for the two isolates of S. lacrymans. Blocks decayed by S. lacrymans isolate Sl 221 had reached an average of 49.4% weight loss after 8 weeks, which corresponded to a percent crystallinity value of approximately 40% at that time. Blocks decayed by S. lacrymans isolate Bb 29 had reached a similar weight loss value (49.9%) by week 12. For these blocks, the percent crystallinity at that time was approximately 37%. These percent crystallinity values were in close agreement, despite the fact that the blocks yielding these results were being decayed by different isolates and reached similar weight loss levels at different times. An alternative explanation for the apparent changes observed in percent crystallinity and average d-spacing may be a re-crystallization of the semi-crystalline material after removal of the hemicelluloses. This would account for both the apparent increase in percent crystallinity and the decrease in average d-spacing. However, this process would likely require a relatively clean separation of the hemicelluloses from the semi-crystalline cellulose chains, which may be difficult to achieve in a biological system. Overall, these results show that the three species of brown rot fungi tested are able to significantly alter wood cellulose crystallinity, as detected by XRD. Most interestingly, it was found that these fungi significantly decrease the average d-spacing of the crystalline planes (on the order of 0.05 Å), as evidenced by the shift of the XRD peak. This phenomenon may support the concept of an elementary fibril arrangement in wood (Ding and Himmel, 2006) as
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Temporal changes in wood crystalline cellulose during degradation by brown rot fungi Caitlin Howell a, *, Anne Christine Steenkjær Hastrup b, Barry Goodell c, Jody Jellison a a
School of Biology and Ecology, University of Maine, 311 Hitchner Hall, Orono, ME 04469, USA Department of Microbiology, University of Copenhagen, Sølvgade 83H, 1307 Copenhagen K, Denmark c Department of Wood Science and Technology, 5755 Nutting Hall, Orono, ME 04469, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2008 Received in revised form 24 November 2008 Accepted 26 November 2008 Available online 11 January 2009
The degradation of wood by brown rot fungi has been studied intensely for many years in order to facilitate the preservation of in-service wood. In this work we used X-ray diffraction to examine changes in wood cellulose crystallinity caused by the brown rot fungi Gloeophyllum trabeum, Coniophora puteana, and two isolates of Serpula lacrymans. All fungi increased apparent percent crystallinity early in the decay process while decreasing total amounts of both crystalline and amorphous material. Data also showed an apparent decrease of approximately 0.05 Å in the average spacing of the crystal planes in all degraded samples after roughly 20% weight loss, as well as a decrease in the average observed relative peak width at 2q ¼ 22.2 . These results may indicate a disruption of the outer most semi-crystalline cellulose chains comprising the wood microfibril. X-ray diffraction analysis of wood subjected to biological attack by fungi may provide insight into degradative processes and wood cellulose structure. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: X-ray diffraction Crystallinity Serpula lacrymans Coniophora puteana Gloeophyllum trabeum Wood decay
1. Introduction Wood cell walls consist of cellulose, hemicelluloses, lignin, pectin, proteins, and other trace materials. Cellulose, which comprises 46–52% of the wood volume in softwoods and 39–52% in hardwoods (Zabel and Morrell, 1992), is composed of linear chains of b-1,4 linked D-anhydroglucopyranose, with cellobiose as the repeating unit (O’Sullivan, 1997). These chains are arranged into elementary fibrils, consisting of 60–70% crystalline cellulose and 30–40% amorphous cellulose, and surrounded by a hemicellulose and lignin matrix (Salmen and Olsson, 1998; Neagu et al., 2006). The crystalline portions of wood are formed when the cellulose chains that comprise the microfibril are close enough to form hydrogen bonds, creating a regularly patterned structure (O’Sullivan, 1997). Recent atomic force microscopy (AFM) work by Ding and Himmel (2006) on corn stover has resulted in a refined model of the arrangement of cellulose chains in the elementary fibril. In this model, the six innermost cellulose chains are the most highly ordered and display an Ib cellulose structure. These chains are surrounded by 12 slightly less crystalline (sub-crystalline) chains,
* Corresponding author. Tel.: þ1 207 581 3032; fax: þ1 207 581 2537. E-mail address: [email protected] (C. Howell). 0964-8305/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2008.11.009
which are surrounded in turn by 18 less ordered (semi-crystalline or non-crystalline) cellulose chains. In wood, hemicelluloses are thought to have a closer association with cellulose than lignin (Salmen, 2004; Lawoko et al., 2005; Neagu et al., 2006). Nevertheless, the exact structure of the cellulose microfibrils, macrofibrils, and the associations of these with hemicelluloses and lignin remain unclear. X-ray diffraction (XRD) has been used for decades as a rapid, non-destructive method for observing the crystalline portion of wood (Cullity, 1978; Cave, 1997; Lichtenegger et al., 1999). The interference pattern created by the wood can be used to determine the overall percent of the wood that is in a crystalline state by comparing the sharp crystalline diffraction signals to the broad signals created by the amorphous material. A number of studies have focused on the modification of crystalline cellulose by brown rot wood decay fungi as characterized by XRD. Those focusing on the depolymerization of cellulose by the brown rot fungus Postia placenta have shown that early in the decay process this fungus will temporarily increase overall crystallinity and the size of the crystallites, presumably due to a preferential degradation of the amorphous regions of the cellulose microfibrils (Highley et al., 1988; Klemen-Leyer et al., 1992). The purpose of this study was to thoroughly examine changes in wood crystallinity caused by three different species of brown rot
C. Howell et al. / International Biodeterioration & Biodegradation 63 (2009) 414–419
fungi over time, and to compare the resulting data to changes observed in weight loss and wood sugar composition. 2. Materials and methods 2.1. Growth conditions Fungi were maintained at 21 C on 2% (w/v) potato dextrose agar (PDA) until inoculation. Fungi used in this study were Serpula lacrymans (Wulfen: Fries) Schro¨ter (1889) Bb 29 from the Technological Institute, Denmark, and Sl 221 provided by Dr H. Kauserud, Norway, Coniophora puteana (Schumach.) P. Karst. (1868) ATCC 44393 and Gloeophyllum trabeum (Pers.:Fr.) Murrill (1908) ATCC 11539. C. puteana and G. trabeum cultures were purchased from the American Type Culture Collection (Manassas, VA, USA). 2.2. Soil block assays Four 1-cm2 pieces of inoculum were taken from the outer edge of mycelium of 3-week-old fungal cultures and placed into modified AWPA soil block jars (AWPA, 2003). Each piece of inoculum was placed at one corner of a pair of birch feeder strips on top of approximately one cup of a 1:1:1 mixture of potting soil, sphagnum peat moss, and horticultural grade vermiculite, hydrated with deionized water. One 1.2 2.5 2.5 cm transverse pine block was placed in the jar after the mycelial mat had covered the set of birch feeder strips. Fungi were allowed to grow for 1, 2, 4, 6, 8, 10, and 12 weeks after the addition of the wood block. There were five replicates per fungus per harvest, as well as five uninoculated controls.
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least-squares peak fitting method with an amorphous standard (O’Sullivan, 1997; Andersson et al., 2003; Thygesen et al., 2005), a Rietveld analysis (Rietveld, 1967, 1969) using the cellulose 1b crystal structure published by Nishiyama et al. (2002), and an area comparison method. No significant differences were found for percent crystallinity calculated by the three methods. Percent crystallinity was calculated from raw XRD spectra by comparing the area of the crystalline regions to the total area. Removal of crystalline material over time was calculated by multiplying the calculated percent crystallinity of a block by the measured dry weight of the block. Removal of amorphous material over time was calculated by multiplying the percent amorphous material (defined as 100%dpercent crystallinity) by the total dry weight of the block. Using this method it was possible to adjust for percent weight loss in calculating the decrease in crystalline and amorphous material over time. Ratio calculations such as percent crystallinity values were not adjusted. Adjusting these values for weight loss would have caused the loss of information and yielded an inaccurate representation of percent crystallinity. 2.5. Wood sugar and Klason lignin analysis Carbohydrate analysis of the ground material of each replicate within a sampling time set was analyzed using high pH anionexchange chromatography with pulsed amperometric detection (HPAEC/PAD) according to Davis (1998). The amount of acidinsoluble lignin in the wood material was analyzed according to the American Wood Preservers’ Association (AWPA) method D1106-96 (AWPA, 1996). 2.6. Statistics
2.3. Processing and X-ray diffraction All blocks were dried for 48 h at 95 C and weighed immediately after harvest. Blocks were ground in a Wiley Mill to pass through a standard 40-mesh screen (420 mm) and pressed into a cylindrical wafer 25 mm in diameter and 4.3-mm thick. Wood wafers were scanned using a Panalytical X’Pert X-Ray Diffraction machine (Panalytical, Netherlands) with symmetric q–2q Bragg–Brentano scattering geometry. Nickel filtered Ka radiation with a wavelength of 1.542 Å was used to perform q–2q scans. The incident beam was passed through a fixed divergence slit of 1/16 and an anti-scatter slit of 1/8 , as well as a 15-mm mask. The diffracted beam was passed through a size 5.0 anti-scatter slit. Data were collected in the 2q-range 5–35 with a step size of 0.01. The scans proceeded at 0.08 per second (150 s per step). 2.4. XRD spectral analysis Due to the variability of published procedures on the analysis of XRD spectra from wood, the spectra from these experiments were processed and analyzed using three different methods: a standard
Statistical analyses of weight losses, percent crystallinity, crystallite size, simple sugar concentrations and d-spacing values were performed employing either one-way or two-way ANOVAs and protected Fisher LSD post hoc tests using SySTAT v.12 (Systat Software Inc., San Jose, CA, USA). 3. Results and discussion 3.1. Weight loss, HPLC and Klason lignin analysis Blocks decayed by all four strains of brown rot fungi showed significant weight losses after 12 weeks. G. trabeum degraded the pine blocks most quickly, reaching a value of 17.2% weight loss after only 2 weeks of decay. Weight losses in blocks decayed by the other fungi tested reached approximately 20% weight loss after 4 weeks (Table 1). Quantification of wood sugars and Klason lignin yielded results consistent with previously published data on wood carbohydrate removal by brown rot fungi, which show hemicellulose depletion up to approximately 20% of weight loss, followed by cellulose
Table 1 Weight loss values for wood blocks inoculated with S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt). The control represents uninoculated wood blocks. Values in parenthesis indicate standard deviations. The asterisks (*) denote unavailable data. Fungal species
Week 1
2
4
6
8
S. lacrymans (Bb) S. lacrymans (Sl) C. puteana (Cp) G. trabeum (Gt) Control (K)
0.7 (1.2) 0.3 (0.1) 0.1 (0.5) 1.8 (0.3) 0.2 (0.4)
2.8 (0.0) 4.1 (0.9) 3.2 (0.9) 17.2 (2.4) 0.0 (0.1)
16.8 (4.8) 21.7 (4.0) 18.4 (2.2) 41.2 (6.2) 0.0 (0.1)
26.3 (5.5) 33.1 (6.7) 25.3 (4.9) 57.7 (1.0) 0.0 (0.0)
32.8 49.4 25.2 59.5 0.0
(7.8) (2.0) (6.8) (2.3) (0.1)
10
12
* 53.8 (3.5) 28.7 (5.5) 56.9 (4.2) *
49.9 (4.5) 58.5 (2.4) 39.5 (11.7) 61.3 (6.1) 0.2 (0.2)
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% of remaining material
S. lacrymans Bb
S. lacrymans Sl
45 40 35 30 25 20 15 10 5 0
wk 0 wk 2 wk 4 wk 6
Glucan
Xylan
45 40 35 30 25 20 15 10 5 0
wk 0 wk 2 wk 4 wk 6
Mannan
Glucan
% of remaining material
G. trabeum wk 0 wk 2 wk 4 wk 6
Xylan
Mannan
C. puteana
45 40 35 30 25 20 15 10 5 0 Glucan
Xylan
45 40 35 30 25 20 15 10 5 0
wk 0 wk 2 wk 4 wk 6
Mannan
Glucan
Xylan
Mannan
Fig. 1. Glucan, xylan and mannan wood sugar analysis data for wood blocks inoculated with S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393, and G. trabeum ATCC 11539 after 0, 2, 4 and 6 weeks.
depletion for the remaining weight loss (Curling et al., 2001). The proportional removal of both hemicellulose and cellulose sugars was consistent for all fungi tested with the exception of C. puteana, which appeared to remove more xylan and mannan and less glucan at comparable weight loss values (Fig. 1). Analysis of Klason lignin levels showed an increasing percent of lignin in the remaining material as the weeks progressed (Table 2), consistent with the known degradation methods of these fungi. 3.2. Percent crystallinity and removal of crystalline and amorphous material over time All fungi showed significant increases in percent crystallinity, or percent of the residual material that was in a crystalline state, compared to the controls as early as week 1 (P < 0.022) (Fig. 2). Percent crystallinity values for the two isolates of S. lacrymans and C. puteana were significantly different by week 4 of decay (P ¼ 0.000), while values for G. trabeum had begun to decline by this point. By week 12, G. trabeum and both S. lacrymans isolates yielded percent crystallinity values lower than the controls, although only G. trabeum was significantly different (P ¼ 0.001). This trend (an increase in crystallinity early in the decay process, followed by a decrease) has been previously observed by other researchers examining wood decayed by fungi (Highley et al., 1988; Klemen-Leyer et al., 1992) and wood treated chemically to extract hemicelluloses (Hult et al., 2003; Åkerholm et al., 2004), and has been attributed to the initial removal of hemicelluloses and other amorphous materials. The observed increase in crystallinity peaks
around week 2 for wood degraded by G. trabeum, and around week 4 for the other fungi as weight losses increase to approximately 20%, a level consistent with the near complete removal of the hemicelluloses (Curling et al., 2001). After week 2 (for G. trabeum) and week 4 (for all other isolates), the percent crystallinity values begin to decrease, possibly due to continuing fungal attack on the crystalline cellulose only after degradation of the more readily available amorphous nutrient sources. Percent crystallinity values for G. trabeum dip below the values for the controls by week 12. This is possibly due to the fact that amorphous lignin is being left behind and comprises a greater percentage of the remaining material as the weeks progress (Table 2). The percent crystallinity values for C. puteana remained above the level of the controls at week 12. This may be due to the relatively low weight loss values achieved by this fungus (39.5%) as compared to the other fungi at this time point. Calculation of the rates of removal of amorphous versus crystalline material revealed that the degradation of the amorphous material began immediately, as confirmed by the HPLC data (Fig. 1). Total amounts of crystalline material began to decrease at week 2 for G. trabeum and week 4 for the other fungi (Fig. 3), again as the samples approached the 20% weight loss benchmark which signals nearly complete degradation of the easily accessible hemicelluloses. The amount of amorphous material appeared to remain constant after week 6 for G. trabeum and after week 8 for S. lacrymans isolate Sl 221, while the relative amount of crystalline material continues to decrease after this point. This may be due to the achievement of a steady state in which the crystalline material is
Table 2 Klason lignin (give in percent of remaining material with standard deviations in parentheses) for wood blocks inoculated with S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt). The control represents uninoculated wood blocks. The asterisks (*) denote unavailable data. Fungal species
S. lacrymans (Bb) S. lacrymans (Sl) C. puteana (Cp) G. trabeum (Gt) Control (K)
Week 1
2
30.5 (0.3) 30.7 (0.4) 30.6 (0.6) 30.1 (1.2) 30.6 (2.1)
30.0 30.8 28.6 33.8 30.0
4 (1.8) (0.7) (0.5) (1.0) (1.4)
35.0 36.7 36.0 48.8 28.8
(1.2) (2.4) (1.0) (3.7) (0.2)
6
8
10
12
39.1 (2.3) 42.7 (3.1) 38.2 (2.5) 61.6 (2.6) 28.5 (0.4)
* 54.4 (2.3) 39.6 (2.3) * *
* 61.0 (3.8) 40.9 (1.6) 64.2 (7.8) 29.1 (0.8)
59.1 (3.1) 66.6 (2.3) 44.0 (6.8) 68.9 (8.7) 28.4 (0.5)
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% Crystallinity
S. lacrymans Bb 55 50 45 40 35 30 25 20 15
K Bb
S. lacrymans Sl
55 50 45 40 35 30 25 20 15
K Sl Week Week Week Week Week Week Week Week 0 1 2 4 6 8 10 12
Week Week Week Week Week Week Week Week 0 1 2 4 6 8 10 12
C. puteana
% Crystallinity
G. trabeum 55 50 45 40 35 30 25 20 15
55 50 45 40 35 30 25 20 15
K Gt
417
K Cp Week Week Week Week Week Week Week Week 0 1 2 4 6 8 10 12
Week Week Week Week Week Week Week Week 0 1 2 4 6 8 10 12
Fig. 2. Percent crystallinity in uninoculated control wood blocks (K, dashed lines), and wood blocks inoculated with S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt) (solid lines).
converted into amorphous material before absorption and ultimate metabolism by the fungi. This conversion seems to occur at or near weight loss levels of 50%. These trends are not observed in wood degraded by S. lacrymans isolate Bb 29 and C. puteana, as these fungi either did not achieve the appropriate weight loss levels early enough in the study period, or at all. 3.3. Average d-spacing Analysis of the peak locations showed a small but consistent and significant rightward shift in the 200 peak in XRD spectra of the wood blocks decayed by fungi, indicating a decrease in the average d-spacing of these crystalline planes. Calculations revealed that the average spacing between the crystalline planes was reduced significantly at week 1 for G. trabeum and C. puteana (P ¼ 0.047 and P ¼ 0.046, respectively) and by week 2 for the two isolates of S.
lacrymans (P ¼ 0.031 (Sl) and P ¼ 0.002 (Bb)). Although the overall change in d-spacing was minimal (approximately 0.05 Å), it was found to be similar for all fungi. This decrease became significant (P ¼ 0.000) at the 20% weight loss plateau in all cases and remained at that level through week 12 of decay (Fig. 4). Slight shifts in the location of the 200 peak in cellulosic material have previously been observed by other researchers, most notably between cellulose from sources classified as Ia-dominant, and those classified as Ib-dominant (Wada et al., 2001). The International Center for Diffraction Data (ICDD) lists the cellulose 200 peak as having a maximum intensity at 21.80 2q for Ia, and at 22.98 for Ib (ICDD, 2008). It is possible, therefore, that the shifts observed in these experiments and attributed to fungal degradation indicate a move toward a more Ib-dominant system. This would be consistent with the model developed by Ding and Himmel (2006) who observed that the more highly ordered chains at the center of
Control Amorphous Decayed Amorphous
Material (g)
2.5
S. lacrymans Bb
Control Crystalline Decayed Crystalline 2.5
2
2
1.5
1.5
1
1
0.5
0.5 0
0 wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
Material (g)
2.5
S. lacrymans Sl
G. trabeum
wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12 2.5
2
2
1.5
1.5
1
1
0.5
0.5
C. puteana
0
0 wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
Fig. 3. Amount of crystalline material (light colors) versus amorphous material (dark colors) in wood blocks degraded by S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt). Grey dotted lines represent values calculated for control blocks harvested at the same time as the inoculated blocks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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S. lacrymans Bb d-spacing ( Å)
8.15
S. lacrymans Sl 8.15
8.1
8.1
8.05
8.05
8
8
7.95
7.95
7.9
K
7.85
wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
G. trabeum
8.15
d-spacing ( Å)
Sl
7.85 wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
C. puteana 8.15
8.1
8.1
8.05
8.05 8
8 7.95
K
7.9
Bb
K
7.95
Gt
7.9
K Cp
7.85
7.9 wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
wk 0 wk 1 wk 2 wk 4 wk 6 wk 8 wk 10 wk 12
Fig. 4. Average d-spacing values between the cellulose crystal planes in uninoculated control wood blocks (K), and wood blocks decayed by S. lacrymans isolate Bb 29 (Bb), S. lacrymans isolate Sl 221 (Sl), C. puteana ATCC 44393 (Cp), and G. trabeum ATCC 11539 (Gt).
the cellulose elementary fibrils display an Ib conformation. The fact that this shift occurs early in the decay process and is constant throughout the remaining weeks may be another indication of the fluidity and availability of the surface chains and, conversely, the rigidity of the more crystalline inner chains. It is interesting to note that significant changes in percent crystallinity and d-spacing, as measured by XRD, are detectable relatively early in the decay process for all fungi tested. Previous studies using wood degraded by G. trabeum have shown that significant decreases in strength properties occur even before significant weight loss is detected (Curling et al., 2001). It has been established that this is most likely due to extensive cellulose and hemicellulose depolymerization (Goodell, 2003). Although this change is most likely to be detectable in the lengthwise direction of the microfibril, it is also possible that the changes in FWHM and 2q values seen in these experiments are a result of the depolymerization process. Also noteworthy is the similarity of the percent crystallinity and weight loss values documented for the two isolates of S. lacrymans. Blocks decayed by S. lacrymans isolate Sl 221 had reached an average of 49.4% weight loss after 8 weeks, which corresponded to a percent crystallinity value of approximately 40% at that time. Blocks decayed by S. lacrymans isolate Bb 29 had reached a similar weight loss value (49.9%) by week 12. For these blocks, the percent crystallinity at that time was approximately 37%. These percent crystallinity values were in close agreement, despite the fact that the blocks yielding these results were being decayed by different isolates and reached similar weight loss levels at different times. An alternative explanation for the apparent changes observed in percent crystallinity and average d-spacing may be a re-crystallization of the semi-crystalline material after removal of the hemicelluloses. This would account for both the apparent increase in percent crystallinity and the decrease in average d-spacing. However, this process would likely require a relatively clean separation of the hemicelluloses from the semi-crystalline cellulose chains, which may be difficult to achieve in a biological system. Overall, these results show that the three species of brown rot fungi tested are able to significantly alter wood cellulose crystallinity, as detected by XRD. Most interestingly, it was found that these fungi significantly decrease the average d-spacing of the crystalline planes (on the order of 0.05 Å), as evidenced by the shift of the XRD peak. This phenomenon may support the concept of an elementary fibril arrangement in wood (Ding and Himmel, 2006) as
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