APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1995, p. 1591–1598 Vol. 61, No. 4 0099-2240/95/$04.0010 Copyright q 1995, American Society for Microbiology Alterations in Structure, Chemistry, and Biodegradability of Grass Lignocellulose Treated with the White Rot Fungi Ceriporiopsis subvermispora and Cyathus stercoreus D. E. AKIN,1* L. L. RIGSBY,1 ANAND SETHURAMAN,2 W. H. MORRISON III,1 2 2 G. R. GAMBLE, AND K.-E. L. ERIKSSON Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia 30604,1 and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 306022 Received 29 September 1994/Accepted 30 January 1995 The white rot fungi Ceriporiopsis subvermispora FP-90031-sp and Cyathus stercoreus ATCC 36910 were evaluated for their ability to delignify Bermuda grass (Cynodon dactylon) stems and improve biodegradability. Compositional and structural alterations in plant cell walls effected by the fungi were determined by nuclear magnetic resonance spectroscopy, gas chromatography of alkali-treated residues, microspectrophotometry, and electron microscopy. Contaminating bacteria and fungi, which grew from unsterilized Bermuda grass stems, did not alter the improvement in grass biodegradability by either of the fungi from that of gas-sterilized stems. The biodegradation of stems by ruminal microorganisms, after treatment for 6 weeks with C. subver- mispora or C. stercoreus, was improved by 29 to 32% and by 63 to 77%, respectively; dry weight losses caused by pretreatment with the fungi were about 20% over that in untreated, control stems. Both fungi preferentially removed aromatics to carbohydrates, and C. subvermispora removed proportionately more guaiacyl units than did C. stercoreus. Substantial amounts of ester-linked p-coumaric and ferulic acids were removed by both fungi, and about 23 and 41% of total aromatics (determined after 4 M NaOH direct treatment) were removed from the plant biomass after incubation with C. subvermispora and C. stercoreus, respectively. UV absorption microspectrophotometry indicated that ester-linked phenolic acids were totally removed from the parenchyma cell walls, and these cells were readily and completely degraded by both fungi. However, aromatic constituents were only partially removed from the more recalcitrant sclerenchyma cell walls, resulting in variation in electron density and random digestion pits after incubation with fiber-degrading bacteria. These fungi varied in their potential to delignify various types of plant cell walls. White rot fungi are the only known organisms that, to any white rot fungi that have been shown to improve the biodeg- extent, mineralize lignin to CO2 and water in pure culture (20), radation of grasses, apparently by specifically attacking various albeit carbon sources such as polysaccharides are required. A aromatic constituents (10, 23). Some work has been done re- considerable amount of information on degradation of various garding their lignin-degrading enzymes and capabilities (29, 31, wood lignins and lignified cell walls is available. Most of the 32), but very little has been done on their attack on specific cell work, including enzymatic studies and molecular biology, has types in grasses. Additional work is needed to fully character- been done with Phanerochaete chrysosporium (17). However, ize and exploit the potential of these fungi, especially with there are many other lignin-degrading white rot fungi, and respect to delignification and modification of grass cell walls. their attributes and abilities to degrade lignin and various plant The objective of this study was to investigate the chemical and cell types differ considerably (2, 3, 17, 27, 30). The complexity structural modifications in plant cell walls effected by these two of the lignified substrate, in addition to the multiple enzymes white rot fungi and the potential of these fungi to upgrade the involved in lignin degradation, impedes research in this area biodegradability of specific cell types in grass stems. (16). The chemistry of grass lignocellulose varies considerably from that of wood. As determined by various extractive pro- MATERIALS AND METHODS cedures, there is less lignin in grasses than in woody plants (33, Microorganisms. Two white rot fungi, C. subvermispora FP-90031-sp (courtesy 34). Grasses contain a high concentration of hydroxycinnamic of R. A. Blanchette, University of Minnesota) and C. stercoreus ATCC 36910 acids as part of the lignocellulosic fraction (21, 24, 33). Anal- (courtesy of K. Karunanandaa, Pennsylvania State University), were used to ysis of wheat stems indicated that p-coumaric and ferulic acids biologically delignify plant biomass. The fungi were maintained on malt extract agar (Difco Laboratories, Detroit, Mich.) or Bermuda grass agar (about 3% are esterified and etherified to other wall components (21). [wt/vol] dried, ground Bermuda grass with 2% [wt/vol] agar). For inoculations, Iiyama et al. (21) proposed that ferulic acid serves as a bridging agar pieces with actively growing nonsporulating hyphae were aseptically placed constituent by being ester linked to polysaccharides and also onto agar plates with plant biomass. Control (untreated) and inoculated plant ether linked to lignin. Lignin extracted from wheat with diox- materials were incubated at 278C. Plant samples. Stem segments from the fourth and fifth internodes below the ane also contained polysaccharides, indicating a close associa- plant apex were collected from 6 to 9 weeks’ regrowth of coastal Bermuda grass tion of aromatics and structural carbohydrates in grasses (22). (Cynodon dactylon) as previously described (10). Cell walls from similar inter- Ceriporiopsis subvermispora and Cyathus stercoreus are two nodes had shown lignin contents (based on extraction with KMnO4) of about 25% (wt/wt) and dry weight losses due to ruminal microorganisms of about 40%. Effect of contaminating microorganisms on activity of white rot fungi. A segment 6-cm long was excised from a single Bermuda grass internode, and six * Corresponding author. Mailing address: Russell Research Center, 1-cm-long sections were cut from the segment and placed into separate vials. Agricultural Research Service, U.S. Department of Agriculture, P.O. Similarly, six 1-cm-long sections were obtained from two additional 6-cm-long Box 5677, Athens, Georgia 30604. internode segments, yielding a total of six vials containing identically matched 1591 1592 AKIN ET AL. APPL.ENVIRON.MICROBIOL. TABLE 1. Dry weight loss and in vitro digestion by ruminal microorganisms of Bermuda grass stems treated with lignin-degrading white rot fungi % Dry weight loss after treatment fora: Sterilization with 2wk 6wk Fungal treatment ethylene oxide Loss due to Loss due to ruminal Loss due to Loss due to ruminal treatment microorganismsb treatment microorganisms None (control) Yes 13.0 6 1.4 A 42.3 6 0.8 A 13.3 6 4.1 A 30.3 6 1.5 A No 20.6 6 4.9 B 42.5 6 2.4 A 21.7 6 4.4 B,C 26.2 6 2.9 A C. subvermispora Yes 19.6 6 5.5 B 38.7 6 8.2 A 33.0 6 1.0 D 40.0 6 3.8 B No 20.3 6 1.2 B 39.8 6 3.4 A 25.4 6 6.0 B 39.3 6 1.4 B C. stercoreus Yes 21.9 6 2.0 B 55.4 6 1.4 B 31.2 6 3.0 C,D 49.4 6 3.0 C No 24.1 6 2.8 B 53.3 6 1.6 B 29.0 6 1.0 B,C,D 49.7 6 1.8 C a Data are averages 6 standard deviations of triplicate replicates (duplicate tubes for each replicate), except for values for C. subvermispora and C. stercoreus (6 weeks), for which two and one values, respectively, were omitted because of no apparent fungal activity. Samples for the two incubations were different, thus leading to variations in values for untreated stems. Values within columns followed by different letters differ at P # 0.05. b Ruminal microbial incubation was for 72 h. groups of three 1-cm-long sections. These six vials constituted replicate 1. A described previously (9). Samples for transmission electron microscopy (TEM) similar process using other stem segments was repeated to yield six vials for were prepared for thin sectioning in Spurr’s epoxy resin as previously described replicate 2 and six vials for replicate 3. These three replicates were used for an (5). A series of TEM micrographs was made at similar magnifications to compare incubation period of 2 weeks. For a 6-week incubation, a different sample set was two representative stems of each treatment, and cell walls were measured with used to provide three additional replicates of six vials each. The sections were digital calipers to assess wall modifications. The wall was measured from lumen freeze-dried overnight, and the dry weights were recorded. Stems in the odd- to lumen, thus including wall positions of two cells plus the middle lamella. For numbered vials were sterilized with ethylene oxide (10), and those in even- light microscopy, samples were prepared in JB-4 Plus as previously described (8); numbered vials were not sterilized. Sections were positioned onto agar plates 16-mm-thick sections were cut for histochemical staining, and 4-mm-thick sec- under aseptic conditions. A pair of plates, i.e., one with sterilized stem sections tions were cut, put on quartz slides, and mounted in glycerin with a quartz and one with nonsterilized sections, within each replicate was left untreated, and coverslip for microspectrophotometry. similar pairs of plates were inoculated with agar pieces containing C. subvermis- Microspectrophotometry. UV absorption microspectrophotometry was car- pora or C. stercoreus. The pairs of plates were sealed in plastic bags for incuba- ried out as previously described (8) on 4-mm-thick sections of Bermuda grass tion. For microscopic study, 3-mm-long sections from one stem only (in order to stems from two incubations with white rot fungi.