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Title: Lignin depolymerization by fungal secretomes and a microbial sink

Abstract

In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining, and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight (M w) by 63% and 75% at pH 7 compared to the M w of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe ( Pseudomonas putida KT2440) was incubated with the fungalmore » secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/methanol/choline (GMC) oxidoreductases and laccases. Altogether, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high lignin content biorefinery stream and that the presence of an aromatic-catabolic bacterium as a 'microbial sink' improves the extent of enzymatic lignin depolymerization.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [2];  [2];  [3];  [3];  [3];  [4];  [5];  [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  3. Consejo Superior de Investigaciones Científicas (CSIC), Madrid (Spain)
  4. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  5. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
OSTI Identifier:
1331971
Report Number(s):
NREL/JA-5100-67108
Journal ID: ISSN 1463-9262
Grant/Contract Number:
AC36-08GO28308
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Green Chemistry
Additional Journal Information:
Journal Volume: 18; Journal Issue: 22; Journal ID: ISSN 1463-9262
Publisher:
Royal Society of Chemistry
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; lignin depolymerization; fungal secretomes; biorefinery

Citation Formats

Salvachua, Davinia, Katahira, Rui, Cleveland, Nicholas S., Khanna, Payal, Resch, Michael G., Black, Brenna A., Purvine, Samuel O., Zink, Erika M., Prieto, Alicia, Martinez, Maria J., Martinez, Angel T., Simmons, Blake A., Gladden, John M., and Beckham, Gregg T. Lignin depolymerization by fungal secretomes and a microbial sink. United States: N. p., 2016. Web. doi:10.1039/C6GC01531J.
Salvachua, Davinia, Katahira, Rui, Cleveland, Nicholas S., Khanna, Payal, Resch, Michael G., Black, Brenna A., Purvine, Samuel O., Zink, Erika M., Prieto, Alicia, Martinez, Maria J., Martinez, Angel T., Simmons, Blake A., Gladden, John M., & Beckham, Gregg T. Lignin depolymerization by fungal secretomes and a microbial sink. United States. doi:10.1039/C6GC01531J.
Salvachua, Davinia, Katahira, Rui, Cleveland, Nicholas S., Khanna, Payal, Resch, Michael G., Black, Brenna A., Purvine, Samuel O., Zink, Erika M., Prieto, Alicia, Martinez, Maria J., Martinez, Angel T., Simmons, Blake A., Gladden, John M., and Beckham, Gregg T. Thu . "Lignin depolymerization by fungal secretomes and a microbial sink". United States. doi:10.1039/C6GC01531J. https://www.osti.gov/servlets/purl/1331971.
@article{osti_1331971,
title = {Lignin depolymerization by fungal secretomes and a microbial sink},
author = {Salvachua, Davinia and Katahira, Rui and Cleveland, Nicholas S. and Khanna, Payal and Resch, Michael G. and Black, Brenna A. and Purvine, Samuel O. and Zink, Erika M. and Prieto, Alicia and Martinez, Maria J. and Martinez, Angel T. and Simmons, Blake A. and Gladden, John M. and Beckham, Gregg T.},
abstractNote = {In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining, and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight (Mw) by 63% and 75% at pH 7 compared to the Mw of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe (Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/methanol/choline (GMC) oxidoreductases and laccases. Altogether, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high lignin content biorefinery stream and that the presence of an aromatic-catabolic bacterium as a 'microbial sink' improves the extent of enzymatic lignin depolymerization.},
doi = {10.1039/C6GC01531J},
journal = {Green Chemistry},
number = 22,
volume = 18,
place = {United States},
year = {Thu Aug 25 00:00:00 EDT 2016},
month = {Thu Aug 25 00:00:00 EDT 2016}
}

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Cited by: 10works
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  • In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining,more » and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight by 63% and 75% at pH 7 compared to the Mw of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe (Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/ methanol/choline (GMC) oxidoreductases and laccases. Overall, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high lignin content biorefinery stream and that the presence of an aromatic-catabolic bacterium as a “microbial sink” improves the extent of enzymatic lignin depolymerization.« less
  • Lignin valorization offers significant potential to enhance the economic viability of lignocellulosic biorefineries. However, because of its heterogeneous and recalcitrant nature, conversion of lignin to value-added coproducts remains a considerable technical challenge. Here, we employ base-catalyzed depolymerization (BCD) using a process-relevant solid lignin stream produced via deacetylation, mechanical refining, and enzymatic hydrolysis to enable biological lignin conversion. BCD was conducted with the solid lignin substrate over a range of temperatures at two NaOH concentrations, and the results demonstrate that the lignin can be partially extracted and saponified at temperatures as low as 60 degrees C. At 120 °C and 2%more » NaOH, the high extent of lignin solubility was accompanied by a considerable decrease in the lignin average molecular weight and the release of lignin-derived monomers including hydroxycinnamic acids. BCD liquors were tested for microbial growth using seven aromatic-catabolizing bacteria and two yeasts. Three organisms (Pseudomonas putida KT2440, Rhodotorula mucilaginosa, and Corynebacterium glutamicum) tolerate high BCD liquor concentrations (up to 90% v/v) and rapidly consume the main lignin-derived monomers, resulting in lignin conversion of up to 15%. Furthermore, as a proof of concept, muconic acid production from a representative lignin BCD liquor was demonstrated with an engineered P. putida KT2440 strain. Our results highlight the potential for a mild lignin depolymerization process to enhance the microbial conversion of solid lignin-rich biorefinery streams.« less
  • Lignin peroxidases (LiPs) are likely catalysts of ligninolysis in many white-rot fungi, because they have the unusual ability to depolymerize the major, recalcitrant, non-phenolic structures of lignin. Some white-rot fungi have been reported to lack LiP when grown on defined medium, but it is not clear whether they exhibit full ligninolytic competence under these conditions. To address this problem, we compared the abilities of a known LiP producer, Phanerochaete chrysosporium, with those of a reported nonproducer, Ceriporiopsis subvermispora, to degrade a synthetic lignin with normal phenolic content, a lignin with all phenolic units blocked, and a dimer, 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol, that representsmore » the major nonphenolic structure in lignin. P. chrysosporium mineralized all three models rapidly in defined medium, but C. subvermispora showed appreciable activity only toward the more labile phenolic compound under these conditions. However, in wood, its natural environment, C. subvermispora mineralized all of the models as rapidly as P. chrysosporium did. Defined media therefore fail to elicit a key component of the ligninolytic system in C. subvermispora. A double-labeling experiment with the dimeric model showed that a LiP-dependent pathway was responsible for at least half of dimer mineralization in wood by P. chrysosporium but was responsible for no more than 6-7% of mineralization by C. subvermispora in wood. Therefore, C. subvermispora has mechanisms for degradation of nonphenolic lignin that are as efficient as those in P. chrysosporium but that do not depend on LiP. 33 refs., 4 figs.« less