Lignin Biodegradation: Microbiology, Chemistry, and Potential Applications

Volume II

T.K. Kirk

• Publisher: CRC Press
• Year: 2019
• ISBN:
  9781351090957
• Edition: 1
• Topic: Biowissenschaften
• Subtopic: Mikrobiologie

Lignin is a generic name for the complex aromatic polymers that are major components of vascular plant tissues. Lignin is abundant; in terms of weight it is probably second only to cellulose among renewable organic materials, and in terms of energy content it might well be the single most abundant. an international seminar on lignin biodegradation was organized and was held May 9 to 11, 1978, at the U.S. Forest Products Laboratory in Madison, Wisconsin. This book records the proceedings of that seminar and is meant to provide a summary of research. Each speaker/author was asked to summarize his research, including his latest unpublished results, and to describe how his work fits into the overall picture. Following two orientation chapters, one a review of lignin structure and morphological distribution in plant cell walls, and the second a review of the microbial catabolism of relevant aromatics, the book is comprised of chapters in the three subject areas given by the book's title. It does, as intended, provide comprehensive coverage of research to date (August 1978).

Chapter 1

Degradation of Lignin and Lignin-Related Substances by Sporotrichum Pul Verulentum (Phanerochaete Chrysosporium)

Paul Ander, Annele Hatakka and Karl-Erik Eriksson

TABLE OF CONTENTS

I. Introduction

II. Degradation of Lignin by S. pulverulentum and Other White Rot Fungi

A. The Influence of Carbohydrates and Cel1obiose:Quinone Oxidoreductase on Lignin Degradation

B. The Importance of Phenol Oxidase Activity in Lignin Degradation

C. Screening of White Rot Fungi for Selective Lignin Degradation

III. Degradation of Vanillic Acid and Related Substances by White Rot Fungi

A. Vanillic Acid Degradation Products

B. On the Formation of Protocathechuic Acid from Vanillic Acid

C. Cultivation of S. pulverulentum in the Presence of Vanillic Acid and Related Substances

1. Mycelium Growth Studies

2. Degradation as Measured by UV and GC Analyses

a. UV Analyses

b. GC Analyses

D. Degradation of Vanillic Acid by Three Other White Rot Fungi

IV. Discussion

V. Summary

Acknowledgments

References

I. Introduction

Next to cellulose, lignin is probably the most common organic compound cycled on earth. Much of this lignin from trees and other plants is converted to humus, thereby importing desirable properties to the soil. Humus influences the structure, aeration, and moisture-holding properties of the soil. It also functions as an ion exchanger and is able to store an release nutrients and carbon dioxide, which can be utilized by growing plants and trees.

Lignin, however, is also a major waste product in pulp manufacture, although large quantities are burned, rather inefficiently, for energy production and recovery of pulping reagents.

With a better understanding of the enzyme mechanisms involved in biological degradation of lignin and lignocellulosic materials, it will be possible to elucidate how lignin is decayed in nature. With this knowledge, it may be possible to use biological delignification processes for technical purposes, as well as to use waste lignins in the production of useful chemicals.¹

II. DEGRADATION OF LIGNIN BY S. pulverulentum AND OTHER WHITE ROT FUNGI

A. The Influence of Carbohydrates and Cellobiose:Quinone Oxidoreductase on Lignin Degradation

For the production of biomechanical pulp (see Volume II, Chapter 14), as well as for delignification of straw and sugar cane bagasse to obtain a better ruminant feed, much research efforts are needed to understand how white rot fungi degrade lignin. One of the first goals in specific fungal degradation of lignin at the Swedish Forest Products Research Laboratory was to obtain mutants of white rot fungi which did not degrade cellulose or hemicellulose, but only degraded lignin. The methods to obtain such mutants were developed by Eriksson and Goodell² and are described in Volume II, Chapter 14.

Somewhat later a new enzyme called cellobiose:quinone oxidoreductase (CBQ) was found in culture solutions Polyporus versicolor and S. pulverulentum.^3,4 This enzyme reduces quiñones produced from phenols by phenol oxidases and simultaneously oxidizes cellobiose to cellobiono-d-lactone. It contains FAD as a prosthetic group and has a molecular weight of 58,000.⁵ The enzyme is induced by cellulose or cellulose degradation products (not by glucose) and seems to be of importance both in cellulose and lignin degradation. This has been further investigated by Ander and Eriksson⁶ with the wild-type and the cellulase- and CBQ-less mutant Cel 44 of S. pulverulentum. Using kraft lignin agar plates, it was found that cellulose was the best cosubstrate for degradation of kraft lignin by the wild-type. The order in which the tested carbohydrates stimulated kraft lignin degradation was as follows: cotton DP 2000 > cotton DP 500 > Walseth cellulose DP 150 > cellobiose or glucose. As an example, glucose and cotton DP 2000 gave 27 and 66% kraft lignin degradation, respectively.⁶ Kirk recently reported that both cellulose and glucose support strong degradation of synthetic ¹⁴C-lignins (dehydrogenative polymerizates [DHP]) (see Volume II, Chapter 4). The reason for this discrepancy in the observations may be that kraft lignin is a partially degraded lignin from the kraft cooking process with a lower molecular weight and with more phenolic hydroxyl groups than DHPs. Addition of glucose or maltose to wood has also been found to stimulate lignin degradation.¹

The importance of CBQ in kraft lignin degradation was further supported by the finding that Cel 44, which does not produce CBQ, could degrade only 20% of the kraft lignin in agar plates in the presence of celulose, as compared to 66% for the wild-type. Some increase in the kraft lignin degradation (from 10 to 20%) by Cel 44 on addition of cellulose is believed to be due to impurities such as glucose and cellobiose in the different cellulose preparations.⁶

Further studies have shown that degradation of kraft lignin, lignin sulfonates, and milled wood lignin by Pleurotus ostreatus was stimulated by cellulose.⁷ Lignin sulfonates were polymerized more in a medium without cellulose compared to a cellulose-containing medium.⁸ This effect is certainly due to the CBQ activity.

At this point, one question seemed particularly important to answer, namely, is CBQ so necessary that a specific degradation of lignin in wood cannot take place in its absence? This question was studied by cultivating Cel 44 on birch, pine, and spruce wood.⁶ As seen in Table 1, lignin was degraded in all three wood species and in birch 31% of the lignin was degraded in 10 weeks without loss of cellulose. The xylan, however, was degraded to a great extent mainly following lignin degradation. Table 1 further shows that the wild-type degrades lignin better than Cel 44 and that birch is easier to degrade than pine and spruce.

All these results indicate that CBQ may be important but not entirely necessary in lignin degradation. CBQ is probably ubiquitous among white rot fungi,⁹ but brown rot fungi do not seem to have this enzyme at all, as investigated with five different species of brown rot fungi.’ The exact function of CBQ is not known at present. It may be part of an extracellular electron transport chain, thereby reducing quiñones to phenols which may be used as substrate for ring-cleaving enzymes. The activity of ring-cleaving enzymes in whte rot fungi is low,” however, and low-molecular-weight phenols may be degraded through other pathways, as described below.

B. The Importance of Phenol Oxidase Activity in Lignin Degradation

By the use of similar methods as in the production of Cel 44, we were able to isolate one phenol oxidaseless mutant (Phe 3) and one phenol oxidase-positive revertant (Rev 9) from S. pulverulentum.¹⁰ As shown in Table 2, the mutant Phe 3 could not degrade lignin or the other wood components. Rev 9, however, degraded all wood components to the same extent as the wild-type. Kraft lignin also was not degraded by Phe 3, unless purified lacease was added to the medium. In that case, degradation of kraft lignin increased to near normal.¹⁰

The lack of wood degradation by Phe 3 may be due to the fact that cellulase and xylanase production was strongly inhibited by the presence of wood phenols (Table 3). By addition of lacease to the Phe 3 cultures, the mutant again produced normal amounts of cellulases. These results may indicate that phenol oxidases have a regulatory role, since lignin as well as polysaccharide degradation is affected by the absence of phenol oxidase production.¹⁰ A phenol oxidaseless (peroxidaseless) mutant of Pha-nerochaete chrysosporium has now also been found (see Volume II, Chapter 5). The mutant does not release ¹⁴CO₂ from labeled DHP.²⁸ These results can all be considered as strong evidence ...

Table of contents

• Cover
• Copyright Page
• Foreword
• The Editors
• Seminar
• Contributors
• Table of Contents
• Chapter 1 Degradation of Lignin and Lignin-Related Substances by Sporotrichum Pul Verulentum (Phanerochaete Chrysosporium)
• Chapter 2 The Role of Laccase in Lignin Biodegradation
• Chapter 3 Enzymatic Transformations of Lignin
• Chapter 4 Physiology of Lignin Metabolism by White-Rot Fungi
• Chapter 5 Genetic and Biochemical Studies on Phanerochaete Chrysosporium and Their Relation to Lignin Degradation
• Chapter 6 Microbial Metabolism of Lignin-Related Aromatics
• Chapter 7 Isolation and Characterization of Lignocellulose-Decomposing Actinomycetes
• Chapter 8 Degradation of Lignin-Related Aromatics and Lignins by Several Pseudomonads
• Chapter 9 Metabolism of Lignin-Related Compounds by Bacteria
• Chapter 10 Biodegradation of Lignin-Related Polystyrenes
• Chapter 11 Microbial Decolorization and Defoaming of Pulping Waste Liquors
• Chapter 12 Regulation and Genetics of The Biodegradation of Lignin Derivatives in Pulp Mill Effluents
• Chapter 13 Lignin Biodegradation and The Production of Ethyl Alcohol from Cellulose
• Chapter 14 Biomechanical Pulping
• Chapter 15 Ultrastructural Changes in The Wall of Spruce Tracheids Degraded by Sporotrichum Pulverulentum
• Chapter 16 Lignin Biodegradation: Summary and Perspectives
• Subject Index
• Chemical Index