(675e) Screening Wood-Decay Fungi for Demethylation of Kraft Lignin Using Two Novel Assays with Applications in Producing Bio-Methanol and Formaldehyde-Based Polymers

Screening
Wood-decay Fungi for Demethylation of Kraft Lignin using Two Novel Assays with
Applications in Producing Bio-Methanol and Formaldehyde-based Polymers

Andrew
Gibson ^1,2, Balaji Venkatesagowda ¹, Iryna Kandybovich ¹,

Aneli M.
Barbosa ¹, Brian Ross ², Lada Malek ², Robert F.H.
Dekker ¹

¹ Biorefining
Research Institute, ² Department of Biology, ³ Northern
Ontario School of Medicine, Lakehead University, Thunder Bay, Ontario, Canada
P7B 5E1

Lignin,
the most abundant aromatic polymer present in the biosphere, is found in the
cell walls of plants rendering the plant resistant to microbial attack and
degradation. This biopolymer has potential applications, yet is currently
considered a waste product (black liquor) in Kraft pulp and paper mills. By
enzymatically modifying Kraft lignin, two immediate products can be obtained: a
modified lignin with a higher phenolic content, and bio-methanol. The first can
be used in formaldehyde-based polymer applications, while the second can be
used in industrial processes and as biofuel. We have developed two specific assay
procedures to screen for enzymatic activity that would produce these products;
selected ion flow tube-mass spectrometry (SIFT-MS) and Ti(III)-nitriloacetic
acid colorimetry. SIFT-MS is a highly sensitive technique capable of measuring
trace amounts of gases including methanol (ppb levels) in the headspace of flask-cultivated
fungi, while the Ti(III)-NTA assay measures the changes in vicinal hydroxyl
groups during the modification of lignin by various fungal species. The Mycological
Herbarium at Lakehead University in conjunction with the Biorefining Research
Institute has a collection of >450 fungal isolates from the Boreal Forest in
northern Canada; 20 of which were screened using these techniques. Fungal cultures
were grown under stationary conditions on a Kraft lignin-minimum salts (Vogel) nutrient
medium for 3 weeks at room temperature. The fungal isolates showed a reduction
of up to 45% of O-methyl content
using the Ti(III)-NTA assay, and a 10-fold increase in methanol concentration as
measured by SIFT-MS compared to controls. The research presents evidence of new enzyme activities that specifically cleave
O-methyl groups from the lignin
molecule. Further work will continue and confirm the existence of these enzyme
activities through their isolation and purification.

Supported by funds from NSERC-CRD
(CRDPJ 38079-09:
Dekker) Canada