Fungi isolated from Miscanthus and sugarcane: biomass conversion, fungal enzymes, and hydrolysis of plant cell wall polymers.

BACKGROUND: Biofuel use is one of many means of addressing global change caused by anthropogenic release of fossil fuel carbon dioxide into Earth's atmosphere. To make a meaningful reduction in fossil fuel use, bioethanol must be produced from the entire plant rather than only its starch or sugars. Enzymes produced by fungi constitute a significant percentage of the cost of bioethanol production from non-starch (i.e., lignocellulosic) components of energy crops and agricultural residues. We, and others, have reasoned that fungi that naturally deconstruct plant walls may provide the best enzymes for bioconversion of energy crops. RESULTS: Previously, we have reported on the isolation of 106 fungi from decaying leaves of Miscanthus and sugarcane (Appl Environ Microbiol 77:5490-504, 2011). Here, we thoroughly analyze 30 of these fungi including those most often found on decaying leaves and stems of these plants, as well as four fungi chosen because they are well-studied for their plant cell wall deconstructing enzymes, for wood decay, or for genetic regulation of plant cell wall deconstruction. We extend our analysis to assess not only their ability over an 8-week period to bioconvert Miscanthus cell walls but also their ability to secrete total protein, to secrete enzymes with the activities of xylanases, exocellulases, endocellulases, and beta-glucosidases, and to remove specific parts of Miscanthus cell walls, that is, glucan, xylan, arabinan, and lignin. CONCLUSION: This study of fungi that bioconvert energy crops is significant because 30 fungi were studied, because the fungi were isolated from decaying energy grasses, because enzyme activity and removal of plant cell wall components were recorded in addition to biomass conversion, and because the study period was 2 months. Each of these factors make our study the most thorough to date, and we discovered fungi that are significantly superior on all counts to the most widely used, industrial bioconversion fungus, Trichoderma reesei. Many of the best fungi that we found are in taxonomic groups that have not been exploited for industrial bioconversion and the cultures are available from the Centraalbureau voor Schimmelcultures in Utrecht, Netherlands, for all to use.

Systematic search for cultivatable fungi that best deconstruct cell walls of Miscanthus and sugarcane in the field.

The goals of our project were to document the diversity and distributions of cultivable fungi associated with decaying Miscanthus and sugarcane plants in nature and to further assess biodegradation of host plant cell walls by these fungi in pure cultures. Late in 2008 and early in 2009 we collected decaying Miscanthus and Saccharum from 8 sites in Illinois and 11 sites in Louisiana, respectively. To recover fungi that truly decay plants and to recover slow-growing fungi, we washed the plant material repeatedly to remove spores and cultivated fungi from plant fragments small enough to harbor at most one mycelium. We randomly selected 950 fungal colonies out of 4,560 microwell colonies and used molecular identification to discover that the most frequently recovered fungal species resided in Hypocreales (Sordariomycetes), Pleosporales (Dothideomycetes), and Chaetothryiales (Eurotiomycetes) and that only a few weedy species were recovered. We were particularly interested in Pleosporales and Chaetothyriales, groups that have not been mined for plant decay fungi. To confirm that we had truly recovered fungi that deconstruct plant cell walls, we assayed the capacity of the fungi to consume whole, alkali-pretreated, ground Miscanthus. Solid substrate cultures of the nine most commonly encountered Ascomycota resulted in Miscanthus weight loss of 8 to 13% over 4 weeks. This is the first systematic, high-throughput, isolation and biodegradation assessment of fungi isolated from decaying bioenergy grasses.

Divergence in mycorrhizal specialization within Hexalectris spicata (Orchidaceae), a nonphotosynthetic desert orchid.

Evidence is accumulating for specialized yet evolutionarily dynamic associations between orchids and their mycorrhizal fungi. However, the frequency of tight mycorrhizal specificity and the phylogenetic scale of changes in specificity within the Orchidaceae are presently unknown. We used microscopic observations and PCR-based methods to address these questions in three taxa of nonphotosynthetic orchids within the Hexalectris spicata complex. Fungal ITS RFLP analysis and sequences of the ITS and nuclear LSU ribosomal gene fragments allowed us to identify the fungi colonizing 25 individuals and 50 roots. Thanatephorus ochraceus (Ceratobasidiaceae) was an occasional colonizer of mycorrhizal roots and nonmycorrhizal rhizomes. Members of the Sebacinaceae were the primary mycorrhizal fungi in every Hexalectris root and were phylogenetically intermixed with ectomycorrhizal taxa. These associates fell into six ITS RFLP types labeled B through G. Types B, C, D, and G were found in samples of H. spicata var. spicata, while only type E was found in H. spicata var. arizonica and only type F was found in H. revoluta. These results provide preliminary evidence for divergence in mycorrhizal specificity between these two closely related orchid taxa. We hypothesize that mycorrhizal interactions have contributed to the evolutionary diversification of the Orchidaceae.