phd thesis on lignin

Lignin is a complex biopolymer abundantly found in all vascular plants. It plays a key role in building connective tissues and giving them strength, rigidity, and resistance to environmental factors such as pathogens. Extracted lignin finds diverse applications in the commercial sector with immense potential in novel value-added applications. Therefore, it is important to develop optimum and sustainable processes for lignin extraction. To this end, one of the aims of the present research was to examine different lignin extraction methods on common wood species present in Newfoundland, Canada – balsam fir, pine, spruce (softwood), birch, maple, and oak (hardwood). Two different lignin extraction methods were studied: (1) the Formacell method, which uses acetic acid/formic acid/water; and (2) the BioEB method, which uses only formic acid/water. Various parameters were tested, including solvent concentration, temperature, cooking time, to determine the most optimal lignin extraction conditions. The results of this study can be applied to inform and improve industrial lignin extraction processes to obtain better yields in the most optimal manner. This thesis also discusses the latest developments in value-added uses of extracted lignin for the preparation of novel bio-based materials. Lastly, it provides a review of the mechanisms of microbial biodegradation of lignin. These microbial ligninolytic mechanisms provide a host of possibilities to overcome the challenges of using harmful chemicals to degrade lignin biowaste in many industries.

Actions (login required)

Downloads per month over the past year

View more statistics

• Skip to main content
• Accessibility information

• Enlighten Enlighten

Enlighten Theses

• Latest Additions
• Browse by Year
• Browse by Subject
• Browse by College/School
• Browse by Author
• Browse by Funder
• Login (Library staff only)

In this section

Lignin conversion to fine chemicals

de Albuquerque Fragoso, Danielle Munick (2018) Lignin conversion to fine chemicals. PhD thesis, University of Glasgow.

The large availability of Kraft lignin as an industrial by-product and its polyaromatic characteristic, is ideal to consider the potential for recycling it into fine chemicals. To depolymerise lignin, solvolysis and hydrogenolysis experiments were performed. This research considered whether the low yields of products (fine chemicals) were related to the low content of β-O-4 bonds or if it was also associated to the dissolution of lignin in the solvent solution employed in the reactions. The type of solvents chosen to check the dissolution effect were those with low cost and were more sustainable than traditional solvents. Water, ethanol, isopropanol (IPA) and acetone were used. The water mixtures were applied in the tests in various proportions (25:75, 50:50, 75:25 solvent/water v:v). Due to their ability to break C-C and C-O bonds in lignin model compounds [1][2], the efficiency of platinum and rhodium in these reactions supported on alumina was also studied. It was found that the non-catalysed (solvolysis) and catalysed reactions showed different selectivities but similar overall yields ~ 10 % wt of monomeric phenols. The difficulty in increasing yields was mainly associated with the highly condensed character of Kraft lignin and re-polymerisation issues. To achieve an understanding of Kraft lignin depolymerisation, isotopic labelling reactions were completed in the presence of deuterated solvents as well as deuterium gas. This gave information on how Kraft lignin depolymerises, the influence of solvent to products formation and the involvement of hydrogen in the rate determining steps in the reactions. These results have led to an initial mechanistic understanding on how this complex molecule may yield alky-phenolic compounds. It was revealed that the solvent was directly involved in the products’ formation and that they were not generated by simple thermolysis. In addition, the presence of catalysts and hydrogen influenced product formation. The compounds showed different kinetic isotopic values, suggesting that each of these molecules came from individual mechanisms, highlighting the complexity of their formation. This was a relevant study as most of lignin depolymerisation mechanistic insights are based on model compounds and not on lignin itself. It was of interest to this project to explore not only different catalysts and their relationship to lignin depolymerisation, but also different lignin types. A simple pre-treatment for lignin extraction using sawdust (from oak and birch wood) in a Parr autoclave reactor in the presence of hydrogen, solvent and high temperature was developed. The lignins obtained after the pre-treatment were named parr-lignin and successfully resulted in polyaromatic molecules with less condensed character compared to lignins from Soda or Kraft pulping. Reactions were carried out with these lignins and a sugar-cane lignin.

Different catalytic systems with these lignins were investigated and how depolymerisation was affected by the metal and support used. The catalysts involved in the reactions included platinum, rhodium, nickel and iron. Various supports such as alumina, zirconia and carbon were tested along with the metals described. It was found that the supports were not inert in these experiments presenting catalytic activity. Materials with low surface area (zirconium catalysts) gave a poor performance compared to the others. In addition, nickel, a non-noble metal, showed as good a catalytic effect in the depolymerisation of these lignins as Pt and Rh. The components in the system influenced the reactions to different extents, especially product distribution. The catalysts had different selectivities and the solvents were not only dissolving lignin but also influencing the results. GPC analysis was performed to give an overview of the condensed level of these lignins and degrees of depolymerisation compared to the original material. GC-MS enabled the identification and quantification of 18 monomeric compounds. The post reaction characterisation of selected alumina catalysts (Pt/Al2O3, Ni/Al2O3 and Al2O3) was performed using XRD, BET, CHN, TPO and Raman Analysis to study the nature of the carbonaceous layer deposited on these materials. The work showed that after reaction the catalysts turned black in colour and the carbon laydown consisted of not only one simple type of carbon, and included graphitic species. The amount of carbon deposited depended on the type of lignin. Oak and birch parr-lignins had the highest and lowest amount of carbon over the catalysts respectively. No obvious trend relating to the type of catalyst, lignin and solvent used to the carbon nature was identified. This work showed that lignins with less condensed nature were less susceptible to solvolysis and more to hydrogenolysis. For example, sugar-cane lignin gave 3.9% of phenolic compounds in the solvolysis while reaction with Rh/Al2O3 gave 12.9% of products. This indicated that more selective cleavage of bonds were promoted by heterogenous catalysts. The results suggested that some compounds were mainly generated via dealkylation and hydrodeoxygenation, allowing a future possibility to generate target molecules. These results were mainly due to the presence of more labile bonds, vulnerable to hydrogenolysis. Highlighting that prior to depolymerisation, the pre-treatment used to extract lignin must be appropriate to avoid depletion of the alkyl-aryl ether bonds (β-O-4 bonds, especially) relevant for fine chemicals generation.

Actions (login required)

Downloads per month over past year

View more statistics

The University of Glasgow is a registered Scottish charity: Registration Number SC004401

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

• View all journals
• Explore content
• About the journal
• Publish with us
• Sign up for alerts
• Open access
• Published: 08 May 2020

Enzymatic and genetic characterization of lignin depolymerization by Streptomyces sp. S6 isolated from a tropical environment

• Fatimah Azizah Riyadi 1   na1 ,
• Analhuda Abdullah Tahir 1   na1 ,
• Nurtasbiyah Yusof 1 ,
• Nurul Syazwani Ahmad Sabri 1 ,
• Megat Johari Megat Mohd Noor 1 ,
• Fazrena Nadia M. D. Akhir 1 ,
• Nor’azizi Othman 2 ,
• Zuriati Zakaria 1 &
• Hirofumi Hara   ORCID: orcid.org/0000-0003-1515-0370 3  

Scientific Reports volume  10 , Article number:  7813 ( 2020 ) Cite this article

7894 Accesses

58 Citations

1 Altmetric

Metrics details

• Applied microbiology
• Environmental biotechnology

The conversion of lignocellulosic biomass into bioethanol or biochemical products requires a crucial pretreatment process to breakdown the recalcitrant lignin structure. This research focuses on the isolation and characterization of a lignin-degrading bacterial strain from a decaying oil palm empty fruit bunch (OPEFB). The isolated strain, identified as S treptomyces sp. S6, grew in a minimal medium with Kraft lignin (KL) as the sole carbon source. Several known ligninolytic enzyme assays were performed, and lignin peroxidase (LiP), laccase (Lac), dye-decolorizing peroxidase (DyP) and aryl-alcohol oxidase (AAO) activities were detected. A 55.3% reduction in the molecular weight (Mw) of KL was observed after 7 days of incubation with Streptomyces sp. S6 based on gel-permeation chromatography (GPC). Gas chromatography-mass spectrometry (GC-MS) also successfully highlighted the production of lignin-derived aromatic compounds, such as 3-methyl-butanoic acid, guaiacol derivatives, and 4,6-dimethyl-dodecane, after treatment of KL with strain S6. Finally, draft genome analysis of Streptomyces sp. S6 also revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin depolymerization, as well as for the mineralization of the lower molecular weight compounds, confirming the lignin degradation capability of the bacterial strain.

Similar content being viewed by others

Novel redox-active enzymes for ligninolytic applications revealed from multiomics analyses of Peniophora sp . CBMAI 1063, a laccase hyper-producer strain

Evaluating lignin degradation under limited oxygen conditions by bacterial isolates from forest soil

Novel bacterial taxa in a minimal lignocellulolytic consortium and their potential for lignin and plastics transformation

Introduction.

Lignocellulosic biomass is the most abundant renewable organic carbon source on earth and can be effectively used as an alternative to biofuels, biochemicals, and biomolecules, with zero net carbon emission. However, the main challenge for its commercial application is the technology for converting lignocellulosic biomass into sugars for bioethanol/biochemicals. Lignocellulosic biomass, the main component of plant structure, mainly consists of two carbohydrate polymers, cellulose and hemicellulose, and an aromatic polymer, lignin, organized and interlinked together. Cellulose and hemicellulose are chains of polysaccharides that can be easily degraded by microbial enzymes or by chemical hydrolysis and are considered the main candidates for bioethanol and biochemical production. Meanwhile, lignin, the most complex among them, is a three-dimensional heterogeneous crosslinked macromolecule comprising numerous aromatic phenylpropanoid monomeric units identified as guaiacyl (G), p-hydroxyphenyl (H), and syringyl (S) units 1 , 2 . In the plant cell wall, lignin acts as a cellular glue between cellulose fibers and is covalently bonded with hemicellulose, thus enhancing its strength and rigidity. In addition, being aromatic in nature and relatively hydrophobic, lignin acts as an antimicrobial and waterproofing agent and provides carbohydrates protection from hydrolysis by microbial enzymes 3 , 4 .

There are several pretreatment approaches that use mechanical, chemical, physicochemical, and biological methods to depolymerize the recalcitrant lignin fraction of lignocellulosic biomass. Among them, biological pretreatment using microbial enzymes appears to be the most promising method, as it offers more environmentally friendly treatment with lower energy requirements and costs. In addition, biological pretreatment could offer advantages, as microbial degradation can be controlled to achieve desired, valuable aromatic and phenolic byproducts, such as vanillin, catechol, styrene, and polyphenols 2 , 5 . Ligninolytic microorganisms attack lignin by forming complex systems of oxidative enzymes 6 . Synergistic actions of groups of extracellular oxidative enzymes are required to initiate lignin depolymerization by generating highly reactive non-specific free radicals, which will simultaneously lead to cleavage of lignin inter-unit bonds (C-C and C-ether bonds) and further breakdown of the lignin structure 2 , 7 .

Some microorganisms, especially white-rot and brown-rot fungi, have been reported to depolymerize lignin during carbon limitation, secreting ligninolytic enzymes, such as lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP) and copper-containing laccase (Lac) 8 , 9 , 10 , 11 . However, most studies have not achieved commercial-scale lignin degradation since fungi have relatively complex genetic and protein expression characteristics 12 . Although less well identified and characterized than the ligninolytic enzymes from fungi, researchers have begun reporting some of the ligninolytic enzymes from bacteria 4 , 13 , 14 , 15 , 16 . However, actual lignin depolymerization and the relationship between the enzymology and molecular understanding of lignin degradation by bacteria are still poorly understood. Bacteria are predicted to possess other classes of ligninolytic enzymes that are not found in fungi. However, these potential ligninolytic bacteria are largely undiscovered, and many novel ligninolytic enzymes may still emerge. In addition, previous characterization studies on lignin-degrading bacteria mostly used lignin model dimers, such as β-aryl ether lignin 12 , veratrylglycerol-β-guaiacol ether 17 , guaiacylglycerol-β-guaiacyl ether 18 , and lignin-related aromatic acids 19 , to evaluate lignin degradation ability. However, depolymerization of polymeric lignin, which can closely resemble natural lignin, is limited and requires further evaluation.

As a tropical country, Malaysia provides a more stable growth temperature for microorganisms compared to temperate countries where the temperature fluctuates year-round. Microbes isolated from the local environment could improve biological treatment processes and further minimize the treatment cost for lignin degradation. In addition, the abundance of oil palm biomass in the country makes it a possible source of bacterial strains with lignin-degrading abilities. Therefore, in this study, biomass from a palm oil mill was used to isolate the lignin-degrading bacterium strain S6. Subsequently, the potential lignin-degrading ability of the strain was evaluated by performing enzymatic assays and gel-permeation chromatography (GPC) using Kraft lignin (KL) as the lignin model compound. The intermediate metabolites of the degraded lignin compounds were also identified using gas chromatography-mass spectrometry (GC-MS). Last, the draft genome sequence of the potential ligninolytic strain was obtained to confirm the degradation process and to reveal all the candidate genes responsible for lignin degradation.

Results and Discussion

Growth of streptomyces sp. s6 on kraft lignin.

To identify the ability of the isolated bacterium to metabolize lignin, the growth of the bacterium on agar plates containing W-minimal media with 2.5 g/L KL was tested. In this study, we utilized KL as the lignin model. Strain S6 was successfully isolated from decaying oil palm empty fruit bunch (OPEFB) and grew well on the tested agar plate. The cells showed a morphology of clear white and leathery spores (Supplementary Fig.  S1 ). To further confirm the ability of the strain to grow on lignin, the growth pattern was observed in liquid culture (in terms of optical density at 600 nm (OD 600 )) using 2.5 g/L KL as the sole carbon source (Supplementary Fig.  S2 ). Although relatively slow, the growth of S6 in the presence of KL was clearly observed. The strain reached a maximum growth after 3 days of incubation, with an OD 600 of approximately 0.2. This showed that despite being recalcitrant, this organism could adapt and metabolize KL as a carbon and energy source. In this study, KL was used as the sole carbon source without additional nutrients, to ensure that the bacterium can degrade lignin substrate. This could be the reason for the slow growth pattern of the bacterium. The organism was expected to depolymerize lignin and take up the lower molecular-weight KL-derived compounds and other organic compounds present in KL to grow.

Previous studies have reported that several lignin-degrading bacteria, including Bacillus ligniniphilus L1 and Streptomyces sp. F2621, require glucose, peptone, or yeast powder as additional carbon and nitrogen sources to stimulate bacterial growth on KL 20 , 21 . According to Zhu et al . 22 , an additional carbon source is necessary for microbial lignin degradation, even though the complete oxidation reaction of lignin is highly exothermic, i.e., generates energy, as the degradation of lignin is still too slow to function as the main source of metabolic energy for the microorganism. However, based on our results, strain S6 was able to grow even when KL was used as the sole carbon source, suggesting that this strain has the ability to degrade and use highly recalcitrant lignin as the carbon source. In addition, in a recent study, the growth of the lignin-degrading bacterium R. opacus was observed on plates after only two weeks of incubation with 2 g/L depolymerized softwood Kraft lignin (DL). R. opacus and P. putida EM42 growth was further evaluated in liquid cultures with 1 g/L DL, and maximum OD 600 values of approximately 0.15 and 0.12, respectively, were observed after two weeks of cultivation, while the maximum OD 600 value for non-depolymerized lignin was approximately one-third of that obtained with DL 23 .

Analyses of extracellular ligninolytic enzyme activities

In most previous studies, lignin degradation by microorganisms were examined by evaluating the activities of ligninolytic enzymes secreted by the organisms. In addition to observing the growth pattern of strain S6, in this study, the activities of various reported ligninolytic enzymes, such as lignin peroxidase (LiP), manganese peroxidase (MnP), dye-decolorizing peroxidase (DyP), laccase (Lac) and aryl-alcohol oxidase (AAO), were also evaluated to confirm the lignin degradation ability of the strain. The cell-free supernatant was used as the crude enzyme to measure the activity of various extracellular ligninolytic enzymes. No MnP activity was detected in strain S6. On the other hand, considerable LiP (7.4 ± 0.032 U/L) and AAO activities (13.0 ± 0.170 U/L) were detected. However, in the case of Lac and DyP, S6 showed a low activity with 0.1 ± 0.095 U/L and 0.1 ± 0.036 U/L, respectively.

Previous enzymatic studies on numerous strong lignin degraders, such as Pandoraea sp. B-6, Comamonas sp. B-9, and Novosphingobium sp. B-7, reported that these strains only secrete MnP and Lac, and no obvious LiP activity was observed when strains were grown on KL 13 , 24 , 25 . In contrast, in this study, the highest enzyme activity was shown for LiP when grown in KL, and no MnP and slight Lac activities were detected. Previous observations for AAO activity were limited, but some fungi, such as Aspergillus nidulans 26 and Pleurotus ostreatus 27 , and bacteria, such as Sphingobacterium sp . ATM 28 , were also reported to show AAO activity. According to Tamboli et al . 28 , AAO catalyzes aryl-α- and α-β-unsaturated γ-alcohol oxidation to the subsequent aldehydes with the simultaneous reduction of O 2 to H 2 O 2 . The H 2 O 2 generated is predicted to be used by ligninolytic peroxidases to regulate lignin degradation and to secrete enzyme activities.

Evaluation of depolymerization activity by GPC analysis

Gel permeation chromatography (GPC) was performed to observe the degradation of the lignin polymer by comparing the changes in the molecular weight distribution of the polymer after being treated with the isolated bacterium. In this study, GPC was used to evaluate the molecular weight (Mw) of KL before and after 7 days of incubation with strain S6. Based on Table  1 , the average Mw of KL on day 0 was approximately 3,401 Da. The Mw of KL decreased to 1,415 Da on day 3 and was further reduced to 1,286 Da after 7 days of being treated with strain S6, achieving 55.3% lignin degradation. In addition, the polydispersity (Mw/Mn) of lignin fragments decreased from 1.5 (control) to 1.1 (day 3 and day 7). A decrease in the molecular weight distribution of treated KL over time indicated that the KL polymer sample was successfully degraded after 7 days of treatment with S6. The bacterial strain showed fast depolymerization of KL after 3 days, as seen from the decrease in the molecular weight of KL and narrowing of the distribution ranges from the chromatograph (see Supplementary Figs.  S3 and S4 ). This result is consistent with our growth curve result that demonstrates a higher growth curve during the first 3 days. Although fast depolymerization was observed after 3 days of treatment, we observed slight depolymerization with the continuation of 7 days of incubation, as seen from the shifted peak in the chromatograph (Supplementary Fig.  S3 ). The presence of these shifted peaks indicates the production of smaller polymers of lignin as a result of bacterial growth. The GPC results suggest that this strain may be able to efficiently degrade polymeric lignin. We believed that strain S6 secretes ligninolytic enzymes that break the intermolecular C-C and C-O bonds of the lignin polymer into oligomers, trimers and dimers of lignin, indicating the feasibility of the biodegradation of the KL polymer. This result is parallel with previous reports on the bacterial utilization and depolymerization of lignin by GPC analysis 29 , 30 , 31 , 32 .

Identification of lignin monomers by GC-MS analysis

GC/MS has been proven to be a very suitable technique to analyze low molecular weight compounds (LMW) released from lignin, as reported by previous research 30 , 31 , 33 , 34 . The LMW compounds produced from KL were analyzed before and after 3 days and 7 days of treatment with strain S6, as summarized in Table  2 and Supplementary Fig.  S5 . Approximately eight aromatic compounds (1,4-dichlorobenzene, 2-methoxyphenol (guaiacol), vanillin, 1,3-dichlorobenzene, apocynin, 2,4-bis(1,1-dimethylethyl) phenol, bis(2-ethylhexyl) phthalate and triphenylphosphine oxide) and nine linear/branched oxygenated hydrocarbons (butyl acetate, 3-methylbutanoic acid, 2-methylbutanoic acid, 3-methyl 2 butanol, 1-4-dichloro benzene, 1-3-dichloro benzene, 2,6-dimethyldecane, 5-methyl-5-propylnonane, 4-methyltridecane, 4,6-dimethyldodecane, and bis(2-ethylhexyl) hexanedioate) were detected by GC-MS analysis. Several lignin monomeric compounds, such as 2,6-dimethyldecane, tridecane, 4-methyl, vanillin and apocynin, that were detected in the untreated KL samples were not detected in the KL samples treated with S6 on days 3 and 7. In addition, new monomer compounds, such as 3-methylbutanoic acid, 2-methylbutanoic acid, 1,4-dichlorobenzene, guaiacol, 5-methyl-5-propylnonane, and 4,6-dimethyldodecane, were detected on day 3 and/or day 7 after treatment with sample S6, suggesting that KL was further degraded by catalytic cleavage of C-C and C-O-C bonds. Since the first step of monomer production from lignin is depolymerization, it is important to break the bonds that hold the phenyl propane units. We believe that strain S6 produces several ligninolytic enzymes, such as peroxidases, that are responsible for depolymerizing lignin polymers and metabolizing low molecular weight aromatic compounds. In addition, the produced lignin fragments may be involved in different reactions, including hydrolysis of C4–ether bonds, dimethoxylation, aromatic ring opening, and C α –C β breakdown 35 . Some linear alkanes, such as 2,6-dimethyldecane and nonane-5-methyl-5-propyl, were also detected, indicating that a ring-opening reaction occurred in strain S6. The phthalate derivative bis(2-ethylhexyl) phthalate has been detected and previously reported from fungal peroxidase degradation of lignosulfonate 36 and from photodegradation of black liquor lignin 37 .

Gene features of Streptomyces sp. S6

The draft genome of strain S6 was 6,420,514 bp in size, with a GC content of 71.23%, 9,405 coding sequences (CDS), 6,064 proteins with predicted functions, and 3,341 hypothetical proteins (Table  3 ). Strain S6 was classified as a gram-positive bacterium, Streptomyces sp ., according to its morphological characteristics, 16S rRNA region, and complete genome sequences. Based on the 16S rRNA sequence phylogenetic tree (Fig.  1 ), strain S6 is closely related to other Streptomyces groups, showing the highest similarity to Streptomyces cavourensis strain NRRL 2740 (NR_043851.1) and Kitasatospora albolonga strain NBRC 13465 (NR_041144.1), a homotypic synonym of Streptomyces albolongus . From the phylogenetic tree, Streptomyces coelicolor A3(2) (NC_003888.3) 38 , Streptomyces sp. F-6 (FJ405358.1) and Streptomyces sp. F-7 (FJ405357.1) 16 , as well as Streptomyces griseorubens strain JSD-1 (KC736485.1) 39 were also previously reported to demonstrate lignin-degrading ability. A gram-positive Bacillus subtilis strain 168 (MH283878.1), was used as the outgroup strain to root the phylogenetic tree. The protein-coding genes from the draft genome sequences of strain S6 were annotated and classified into 23 and 27 functional classes/subsystems according to COG and RAST, respectively. Based on the COG functional categories (Supplementary Table  S1 ), general processes and metabolic pathways related to amino acids, carbohydrates, fatty acids, and lipids were dominant. In addition, the moderate quantity of categories, such as secondary metabolite biosynthesis, transport, and catabolism in the subsystem features suggests that strain S6 is capable of surviving and metabolizing lignin or aromatic compounds.

Phylogenetic analysis of Streptomyces strain S6 (highlighted) with the related species strains based on 16S rRNA gene homology. This tree was generated by the maximum-likelihood algorithm using Jukes-Cantor distance correction and the bootstrap resampling method after 500 replications, which was conducted with MEGA 7 software. GenBank accession numbers of related species are shown in parentheses. Bacillus subtilis strain 168 (MH283878.1) was used as an outgroup to root the tree.

Identification of ligninolytic enzyme genes through the draft genome sequence of strain S6

Lignin degradation that occurs in nature is mainly a result of two processes: first is the depolymerization of native polymeric lignin to produce low molecular weight aromatic compounds, followed by the mineralization of the resulting aromatics. Depolymerization of native lignin is driven by extracellular oxidative enzymes, such as Lip, MnP, and Lac, which have been highly reported in fungi. Bacteria are thought to have a lesser amount of these powerful ligninolytic enzymes and are generally predicted to play a key role in the second stage of lignin degradation: the mineralization of lignin-derived aromatic compounds. Although studies on the enzymology of lignin-degrading bacteria are still limited, bacteria are also expected to use extracellular peroxidases to initiate lignin depolymerization, and these enzymes are still used as indicators of bacterial lignin degradation. The draft genome of strain S6 was used to verify the presence of genes that encode known ligninolytic enzymes. Since no MnP activity was detected in this study, it was not surprising that searching for the MnP gene in strain S6 also showed the absence of the gene. However, when comparing strain S6 with the reference genome that encodes LiP and Lac, only open reading frames (ORFs) with very low amino acid identities were found: ~32% against LPOA of Phanerochaete chrysosporium (UniProt Entry: P06181) and ~34% against lccA of Haloferax volcanii strain ATCC 29605 (UniProt Entry: D4GPK6).

The absence or low enzymatic activities detected for some of the enzymes could be due to the inadequacy of the reaction conditions and substrate concentration used in the reported enzymatic assays 40 , 41 , 42 , 43 ; therefore, further optimization is required in the next studies. However, in a previous study, Shi et al . 44 also reported that although Lac and MnP activities were detected for Cupriavidus basilensis B-8, no MnP or Lac genes were found in the strain. According to Davis et al . 45 , homologs of the most common ligninolytic peroxidases, such as LiP and MnP, have not been fully acquired in biochemical studies on lignin-degrading bacteria, and analyzing the gene sequences or proteomes of ligninolytic bacteria revealed no homologs. In addition, previous studies on lignin degradation were mostly conducted on fungi. Thus, the well-known ligninolytic peroxidases, the Class II plant peroxidase superfamily, are only limited to fungi, but bacteria are expected to possess unique lignin-degrading mechanisms and other types of peroxidases 46 . Surprisingly, our analysis showed that some genes in S6 demonstrated high homology and similarity with genes encoding DyP in the reference genome, with 59% similarity to the DyP of Rhodococcus jostii RHA1 (UniProt Entry: Q0SE24) and 44% similarity to the Tfu_3078 gene of Thermobifida fusca strain YX (UniProt Entry: Q47KB1). These results are consistent with the DyP activity detected, although at low quantity. Variations in the genes and activities of enzymes secreted by S6 in comparison to previous reports could indicate that strain S6 possesses a diverse regulation mechanism for novel ligninolytic enzyme-encoding genes.

Putative genes responsible for lignin degradation and central intermediate metabolic pathways based on draft genome sequences

The complex structure of lignin requires the synergistic action of various lignin-degrading enzymes. Exploring the draft genome of strain S6 revealed the presence of putative genes that could be responsible for the degradation of lignin and lignin-derived aromatic compounds (Supplementary Table  S2 ). Peroxidases are reported to be the key enzymes in lignin degradation and could be involved in the initial depolymerization stage 47 , 48 . The draft genome of strain S6 showed the presence of bacterial peroxidases, such as catalase, catalase-peroxidase, peroxiredoxins, glutathione peroxidase, and DyP-type/ deferrochelatase peroxidase, which may be responsible for the enzymatic activities observed during the peroxidase assay. In a previous study on bacterial lignin degradation, DyP appeared to have wide substrate specificity and was shown to degrade not only high redox anthraquinone dyes and aromatic sulfides 49 but also veratryl alcohol, phenolic/nonphenolic lignin compound units 9 , 17 , and manganese 17 . In addition, DyP was also reported to degrade the aryl ether bonds in lignin model compounds 12 , 50 . Catalase peroxidase was reported to degrade a phenolic lignin model compound 46 . Peroxiredoxin is a cysteine-dependent peroxidase that reacts with hydrogen peroxide, organic hydroperoxides, or peroxynitrite. Peroxiredoxin is critical in bacterial cells and acts as an antioxidant defense system to protect cellular components from oxidative damage while regulating various signaling processes, such as cell proliferation, reactive oxygen species scavenging, and cell death 51 .

Several oxidoreductases, ferroxidases (including multicopper oxidases), oxidases, reductases, and dehydrogenases were also present in the genome of strain S6. These enzymes might also be responsible for the AAO activity of strain S6. The oxidases function as auxiliary enzymes by generating hydrogen peroxides utilized by peroxidases for the degradation of lignin and aromatics 52 . The oxidoreductases degrade lignin and aromatic compounds by generating nonspecific free radicals and reactive intermediates. Thus, for cell survival, these intermediates need to be removed or transformed into more stable and less toxic compounds 53 . In addition, multicopper oxidase, which was reported as a bacterial laccase, was also detected. Thus, the laccase activity observed in this study could be due to the presence of this enzyme. Multicopper oxidase functions by reducing dioxygen to water while oxidizing phenolic and nonphenolic compounds to their respective radical species, which can undergo further hydration, oxidation, and polymerization/depolymerization reactions 54 , 55 . Dehydrogenase works in the lignin mineralization stage by cleaving ether linkages and targeting toxic aldehydes, converting them into stable intermediates intracellularly 56 . According to Sato et al . 57 , alcohol dehydrogenase, when combined with short-chain dehydrogenases/reductases and glutathione S-transferase, can synergistically degrade ether linkages of the lignin model compound.

The draft genome analysis of strain S6 also revealed several proteins related to the oxidative stress response for protection from reactive species and detoxification mechanisms during aromatic metabolism, including catalase, superoxide dismutase, glutathione, glutaredoxins, peroxiredoxins, and thioredoxin. Cytochrome P450-related genes, a superfamily of heme-thiolate proteins, were also detected in the draft genome of S6. Cytochrome P450 detected in S6 was also previously reported to support lignin degradation by acting as an oxidase enzyme and was also found to catalyze several enzymatic reactions for the conversion of aromatic/xenobiotic chemicals into more polar and/or less toxic derivatives 58 , 59 . In addition, lignin degradation by fungi requires the support of the quinone oxidoreductase system to utilize Fenton chemistry for degradation 60 . Thus, the presence of quinone oxidoreductase genes in strain S6 could also indicate its lignin-degrading ability. In previous reports, NADPH:quinone oxidoreductase was expressed by the bacterium Pandoraea ISTKB and fungus Trametes versicolor , and the gene was reported to degrade lignin by the Fenton reaction 61 , 62 . Some transferase and hydratase enzymes, such as acetyl-CoA acetyltransferase and enoyl-CoA hydratase, were also reported to have roles in the degradation of the aromatic compound benzoate 63 .

Many studies have reported that bacteria take part in the mineralization of lignin-derived low-molecular-weight aromatic compounds. These aromatic compounds generally have restricted chemical reactivity and are commonly attacked with the help of oxygen by oxygenase enzymes 64 , 65 . Such limited reactivities also lead to the production of some common central intermediates, such as catechol, protocatechuate, gentisate or homogentisate, that will further undergo central-ring cleavage catalyzed by ring-cleaving dioxygenase. Detection of these metabolic pathways in strain S6 confirmed the potential of this strain for lignin degradation. Metabolic pathway analysis with the RAST subsystem and KEGG revealed the genes involved in the metabolism of central aromatic intermediates (Supplementary Table  S3 ). The draft genome of S6 revealed genes responsible for the catechol branch of the beta-ketoadipate and homogentisate pathways as well as salicylate and gentisate catabolism, including catechol 2,3-dioxygenase, homogentisate 1,2-dioxygenase, 4-hydroxyphenylpyruvate dioxygenase, and fumarylacetoacetase, which are involved in the degradation of central intermediate pathways.

Materials and Methods

Bacterial growth on kraft lignin.

Sample preparation and lignin-degrading bacterial isolation were performed according to Tahir et al . 66 . Isolated bacteria were grown in 100 mL of Luria Bertani (LB) broth for cell enrichment and incubated at 30 °C with shaking at 160 rpm until OD 600 ~ 1.0 was reached. After that, a 50 mL aliquot of each bacterial strain was centrifuged at 10,000 rpm for 10 min to pellet the cells. The bacterial cells were washed twice with W-minimal media to remove LB broth completely and resuspended in 50 mL of W-minimal media to be used as a seed culture for KL degradation. The washed cells were inoculated into W-minimal media with KL at the initial OD 600 ∼ 0.1 in a 150 mL flask and incubated at 30 °C and 160 rpm for 10 days. KL was used as the sole carbon source. The medium without seed culture was used as a control. One milliliter of the sample was taken daily to evaluate bacterial growth. Lignin is oxidized during the growth conditions, and the color of KL is dark, which could considerably interfere with the absorbance measurements. Thus, the culture sample was centrifuged, and the obtained cells were diluted with W-minimal media, vortexed and used to measure the OD 600 .

Ligninolytic enzyme activity

Collected samples were centrifuged at 10,000 rpm for 5 min to separate the supernatant from the cell pellets. The cell-free supernatant was collected and used as a crude enzyme to measure ligninolytic enzyme activity. LiP enzyme activity was measured following veratryl (3,4-dimethoxybenzyl) alcohol oxidation to veratryl aldehyde 40 . The enzyme reaction consisted of 500 µL of 100 mM sodium tartrate buffer (pH 3.8), 500 µL of 4 mM veratryl alcohol, and 0.1 mL of crude enzyme. To start the reaction, 0.1 mL of H 2 O 2 (2 mM) was added to the mixture, and incubated at 30 °C for 5 min. A change in absorbance at 310 nm (ɛ = 9,300 M/cm) was observed. MnP activity was determined based on phenol red assay 41 by mixing 0.0025% phenol red, 50 mM sodium tartrate buffer (pH 4.5), 0.2 mM MnSO 4 , 0.1 mM H 2 O 2 , and crude enzyme in a total volume of 1 mL. The enzyme mixture was incubated at 30 °C for 5 min and monitored at 431 nm (ɛ = 22,000 M/cm). Lac enzyme activity was assayed through guaiacol oxidation 42 . The enzymatic mixture consisted of 2 mM guaiacol, 10 mM acetate buffer (pH 5.0), and crude enzyme to a total volume of 1 mL. Guaiacol oxidation was determined by incubating the mixture at 25 °C for 2 hours, and the increase in the absorbance at 450 nm (ɛ = 12,100 M/cm) was observed. DyP activity was determined by monitoring the anthraquinone dye decolorization 43 Reactive Black 5 (RB5) at 597 nm (ɛ = 37,000 M/cm). The enzymatic reaction consisted of 25 mM citrate buffer (pH 3) and crude enzyme at a pH of 3.2, followed by the addition of 119 µM RB5. The reaction was started by adding 0.3 mM H 2 O 2 at 30 °C. The lignin-degrading auxiliary enzyme activity of AAO was also determined by observing veratryl (3,4-dimethoxybenzyl) alcohol oxidation to veratryl aldehyde at 310 nm (ɛ = 9,300 M −1 cm −1 ) 67 . The enzymatic reaction consisted of 5 mM veratryl alcohol, 0.1 M sodium phosphate buffer (pH 6.0), and crude enzyme. All enzymatic assays were conducted in triplicate.

Characterization of lignin-degrading activity by GPC and GC-MS analysis

The KL molecular weight (Mw) distribution before and after pretreatment with strain S6 was determined using aqueous GPC (LC-20AD GPC Shimadzu, Japan), and the procedure was performed using the acetobromination method 68 and following the method by Tahir et al . 66 . Briefly, tetrahydrofuran (THF) (HPLC grade, without stabilizer) was used as the mobile phase, and Styragel HR-5E, as well as Styragel HR-1 (Waters, Milford, MA, USA), were used as the separation columns. System calibration was performed using several polystyrene standards (Supplementary Fig.  S4 ) and detected by UV at a wavelength of 280 nm. Each analysis was performed in triplicate. The degradation products were detected using GC-MS analysis according to Tahir et al . 66 . Briefly, once the culture in LB medium reached OD 600 ~ 1.0, the cells were harvested by centrifugation at 10,000 × g for 10 min and resuspended twice in 100 mL of W-minimal media, prior to washing. Then, 10 mL of W-minimal media and 2.5 g/L KL were added to the cells for seven days, and the cells were incubated at 30 °C with shaking at 160 rpm. Aliquots (1 mL) of each culture were collected every 24 hours and used as the sample. The identification of intermediate metabolites of lignin-derived compounds from the isolate was performed by comparing the mass spectra to the National Institute of Standards and Technology 11 (NIST 11) library provided with the instrument.

Draft genome sequencing and functional annotation

The genomic DNA of S6 was extracted prior to draft genome sequencing using the QIAamp DNA Mini Kit (Qiagen). The draft genomic libraries were sequenced on the Ion X5 XL sequencer system (Thermo Fisher Scientific). Reads from the Ion X5 XL sequencer were quality trimmed using CLC Genomics Workbench software (version 11.0.1; CLC bio, Aarhus, Denmark). High-quality reads were de novo assembled using the same software. The sequences and homologous sequences obtained from GenBank were aligned with the Clustal W 2.0 algorithm. Phylogenetic analysis was performed with the maximum-likelihood algorithm using Jukes-Cantor distance correction and the bootstrap resampling method from the software package MEGA 7 69 . The 16S rRNA gene sequence of Bacillus subtilis strain 168 (MH283878.1), taken from GenBank, was used as an outgroup to root the tree. The genome sequences of bacterial strains were functionally annotated using the Rapid Annotation using Subsystem Technology (RAST) database. Protein-coding genes were also annotated and grouped based on the functional classes using the Cluster of Orthologous Groups (COG) and PATRIC databases. Genes related to lignin degradation in strain S6 were identified using the KEGG database and manually annotated by performing a BLASTP search against the ‘nr’ database. Genes involved in the metabolism of central aromatic intermediates were identified using the RAST subsystem features.

This study demonstrated that a Streptomyces sp. isolated from a tropical environment could be a useful choice for lignin degradation. The strain was successfully proven to possess KL-degrading ability, while producing various low molecular weight lignin-derived compounds, and significant enzymatic activities (LIP, LAC and AAO). Comprehensive draft genomic analysis of Streptomyces sp. S6 revealed various candidate genes related to lignin degradation and potential degradation pathways. To fully understand the metabolic characteristics of lignin and to identify the actual enzymes that are responsible for lignin degradation by strain S6, more experiments and investigations, such as gene expression and proteomics, are needed in the future. Since the bacterial lignin depolymerization mechanism has not been fully understood, a study that combines both characterization and genomic assays using KL as the lignin model is highly important and can have an impact on the conversion of lignin into renewable chemicals.

Data availability

All data generated or analyzed during this study are included in this published article (and the Supplementary Information files). The Whole Genome Shotgun (WGS) project used in this paper has been superseded by the complete genome in (non-WGS) GenBank record CP040654 ( https://www.ncbi.nlm.nih.gov/nuccore/CP040654 ). The version described in this paper is version SDIJ01000000, which consists of sequences SDIJ01000001-SDIJ01003896.

Isikgor, F. H. & Becer, C. R. Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers. Polym. Chem. 6 , 4497–4559 (2015).

Article   CAS   Google Scholar  

Tian, X., Fang, Z., Smith, R. L., Wu, Z. & Liu, M. Properties, Chemical Characteristics and Application of Lignin and Its Derivatives in Production of Biofuels and Chemicals from Lignin. Production of Biofuels and Chemicals from Lignin (eds. Fang, Z. & L. Smith, R.) 3–33 (Springer, 2016).

Hatti-kaul, R. & Ibrahim, V. Lignin-degrading enzymes: an overview in Bioprocessing Technologies in Biorefinery for Sustainable Production of Fuels, Chemicals, and Polymers (eds. Yang, S. T., El-Enshasy, H., Thongchul, N.) 167–192 (Wiley, 2013).

Olajuyigbe, F. M., Fatokun, C. O. & Oyelere, O. M. Biodelignification of some agro-residues by Stenotrophomonas sp. CFB-09 and enhanced production of ligninolytic enzymes. Biocatal. Agric. Biotechnol. 15 , 120–130 (2018).

Article   Google Scholar  

Bugg, T. D. H. & Rahmanpour, R. Enzymatic conversion of lignin into renewable chemicals. Curr. Opin. Chem. Biol. 29 , 10–17 (2015).

Article   CAS   PubMed   Google Scholar  

Vares, T., Kalsi, M. & Hatakka, A. Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw. Appl. Environ. Microbiol. 61 , 3515–3520 (1995).

Article   CAS   PubMed   PubMed Central   Google Scholar  

Levasseur, A. et al . FOLy: An integrated database for the classification and functional annotation of fungal oxidoreductases potentially involved in the degradation of lignin and related aromatic compounds. Fungal Genet. Biol. 45 , 638–645 (2008).

Barrasa, J. M., Camarero, S., Martinez, A. T. & Ruel, K. Ultrastructural aspects of wheat straw degradation by Phanerochaete chrysosporium and Trametes versicolor. Appl. Microbiol. Biotechnol. 43 , 766–770 (1995).

Salvachúa, D., Prieto, A., Martínez, Á. T. & Martínez, M. J. Characterization of a novel dye-decolorizing peroxidase (DyP)-type enzyme from Irpex lacteus and its application in enzymatic hydrolysis of wheat straw. Appl. Environ. Microbiol. 79 , 4316–4324 (2013).

Article   PubMed   PubMed Central   CAS   Google Scholar  

Salame, T. M. et al . Inactivation of a Pleurotus ostreatus versatile peroxidase-encoding gene (mnp2) results in reduced lignin degradation. Environ. Microbiol. 16 , 265–277 (2014).

Soden, D. M., O’callaghan, J. & Dobson, A. D. W. Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiology 1526 , 40–4003 (2018).

Google Scholar  

Ahmad, M. et al . Identification of DypB from Rhodococcus jostii RHA1 as a lignin peroxidase. Biochemistry 50 , 5096–5107 (2011).

Shi, Y. et al . Biochemical investigation of kraft lignin degradation by Pandoraea sp. B-6 isolated from bamboo slips. Bioprocess Biosyst. Eng. 36 , 1957–1965 (2013).

Xu, Z., Qin, L., Cai, M., Hua, W. & Jin, M. Biodegradation of kraft lignin by newly isolated Klebsiella pneumoniae, Pseudomonas putida, and Ochrobactrum tritici strains. Environ. Sci. Pollut. Res. 25 , 14171–14181 (2018).

Singh, R. et al . Improved manganese-oxidizing activity of DypB, a peroxidase from a lignolytic bacterium. ACS Chem. Biol. 8 , 700–706 (2013).

Yang, Y. S., Zhou, J. T., Lu, H., Yuan, Y. L. & Zhao, L. H. Isolation and characterization of Streptomyces spp. strains F-6 and F-7 capable of decomposing alkali lignin. Environ. Technol. 33 , 2603–2609 (2012).

Brown, M. E., Barros, T. & Chang, M. C. Y. Identification and Characterization of a Multifunctional Dye Peroxidase from a Lignin-Reactive Bacterium. ACS Chem. Biol. 7 , 2074–2081 (2012).

Ghatge, S., Yang, Y., Song, W. Y., Kim, T. Y. & Hur, H. G. A novel laccase from thermoalkaliphilic bacterium Caldalkalibacillus thermarum strain TA2.A1 able to catalyze dimerization of a lignin model compound. Appl. Microbiol. Biotechnol. 102 , 4075–4086 (2018).

Nishimura, M., Ooi, O. & Davies, J. Isolation and characterization of Streptomyces sp. NL15-2K capable of degrading lignin-related aromatic compounds. J. Biosci. Bioeng. 102 , 124–127 (2006).

Raj, A., Reddy, M. M. K., Chandra, R., Purohit, H. J. & Kapley, A. Biodegradation of kraft-lignin by Bacillus sp. isolated from sludge of pulp and paper mill. Biodegradation 18 , 783–792 (2007).

Tuncer, M., Kuru, A., Isikli, M., Sahin, N. & Celenk, F. G. Optimization of extracellular endoxylanase, endoglucanase and peroxidase production by Streptomyces sp. F2621 isolated in Turkey. J. Appl. Microbiol. 97 , 783–791 (2004).

Zhu, D. et al . Biodegradation of alkaline lignin by Bacillus ligniniphilus L1. Biotechnol. Biofuels 10 , 44 (2017).

Ravi, K. et al . Bacterial conversion of depolymerized Kraft lignin. Biotechnol. Biofuels 12 , 56 (2019).

Article   PubMed   PubMed Central   Google Scholar  

Chen, Y. H. et al . Biodegradation of kraft lignin by a bacterial strain Comamonas sp. B-9 isolated from eroded bamboo slips. J. Appl. Microbiol. 112 , 900–906 (2012).

Chai, L. Y. et al . Depolymerization and decolorization of kraft lignin by bacterium Comamonas sp. B-9. Appl. Microbiol. Biotechnol. 98 , 1907–1912 (2014).

Varela, E., Guillén, F., Martínez, A. T. & Martínez, M. J. Expression of Pleurotus eryngii aryl-alcohol oxidase in Aspergillus nidulans: purification and characterization of the recombinant enzyme. Biochim. Biophys. Acta 1546 , 107–13 (2001).

Okamoto, K. & Yanase, H. Aryl alcohol oxidases from the white-rot basidiomycete Pleurotus ostreatus. Mycoscience 43 , 391–395 (2002).

Tamboli, D. P., Telke, A. A., Dawkar, V. V., Jadhav, S. B. & Govindwar, S. P. Purification and characterization of bacterial aryl alcohol oxidase from Sphingobacterium sp. ATM and its uses in textile dye decolorization. Biotechnol. Bioprocess Eng. 16 , 661–668 (2011).

Azman, N. F. et al . Depolymerization of lignocellulose of oil palm empty fruit bunch by thermophilic microorganisms from tropical climate. Bioresour. Technol. 279 , 174–180 (2019).

Mathews, S. L., Grunden, A. M. & Pawlak, J. Degradation of lignocellulose and lignin by Paenibacillus glucanolyticus. Int. Biodeterior. Biodegrad. 110 , 79–86 (2016).

Shi, Y. et al . Directed bioconversion of Kraft lignin to polyhydroxyalkanoate by Cupriavidus basilensis B-8 without any pretreatment. Process Biochem. 52 , 238–242 (2017).

Yang, C.-X., Wang, T., Gao, L.-N., Yin, H.-J. & Lü, X. Isolation, identification and characterization of lignin-degrading bacteria from Qinling, China. J. Appl. Microbiol. 123 , 1447–1460 (2017).

Chen, Y. et al . Application of Fenton pretreatment on the degradation of rice straw by mixed culture of Phanerochaete chrysosporium and Aspergillus niger. Ind. Crops Prod. 112 , 290–295 (2018).

Raj, A., Krishna Reddy, M. M. & Chandra, R. Identification of low molecular weight aromatic compounds by gas chromatography–mass spectrometry (GC–MS) from kraft lignin degradation by three Bacillus sp. Int. Biodeterior. Biodegradation 59 , 292–296 (2007).

Zhao, Y. Sustainable Aromatics: Synthesis and Hydrogenolysis of Lignin Monomer Compounds. PhD thesis, University of Leeds (2014).

Shin, K.-S. & Lee, Y.-J. Depolymerisation of lignosulfonate by peroxidase of the white-rot basidiomycete, Pleurotus ostreatus. Biotechnol. Lett. 21 , 585–588 (1999).

Ksibi, M. et al . Photodegradation of lignin from black liquor using a UV/TiO2 system. J. Photochem. Photobiol. A Chem. 154 , 211–218 (2003).

Majumdar, S. et al . Roles of small laccases from Streptomyces in lignin degradation. Biochemistry 53 , 4047–4058 (2014).

Feng, H. et al . Lignocellulose degradation by the isolate of Streptomyces griseorubens JSD-1. Funct. Integr. Genomics 15 , 163–173 (2015).

Camarero, S., Sarkar, S., Ruiz-Dueñas, F. J., Martínez, M. J. & Martínez, A. T. Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites. J. Biol. Chem. 274 , 10324–30 (1999).

Archibald, F. S. A new assay for lignin-type peroxidases employing the dye Azure B. Appl. Environ. Microbiol. 58 , 3110–3116 (1992).

Arora, D. S. & Sandhu, D. K. Laccase production and wood degradation by a white-rot fungus Daedalea flavida. Enzyme Microb. Technol. 7 , 405–408 (1985).

Kim, S. J. & Shoda, M. Purification and characterization of a novel peroxidase from Geotrichum candidum dec 1 involved in decolorization of dyes. Appl. Environ. Microbiol. 65 , 1029–35 (1999).

Shi, Y. et al . Characterization and genomic analysis of kraft lignin biodegradation by the beta-proteobacterium Cupriavidus basilensis B-8. Biotechnol. Biofuels 6 (2013).

Davis, J. R. et al . Genome Sequence of Streptomyces viridosporus Strain T7A ATCC 39115, a Lignin-Degrading Actinomycete. Genome Announc. 1 , 1–2 (2013).

de Gonzalo, G., Colpa, D. I., Habib, M. H. M. & Fraaije, M. W. Bacterial enzymes involved in lignin degradation. J. Biotechnol. 236 , 110–119 (2016).

Article   PubMed   CAS   Google Scholar  

Bugg, T. D. H., Ahmad, M., Hardiman, E. M. & Singh, R. The emerging role for bacteria in lignin degradation and bio-product formation. Curr. Opin. Biotechnol. 22 , 394–400 (2011).

Brown, M. E. & Chang, M. C. Y. Exploring bacterial lignin degradation. Curr. Opin. Chem. Biol. 19 , 1–7 (2014).

Colpa, D. I., Fraaije, M. W. & van Bloois, E. DyP-type peroxidases: a promising and versatile class of enzymes. J. Ind. Microbiol. Biotechnol. 41 , 1–7 (2014).

Rahmanpour, R. & Bugg, T. D. H. Characterisation of Dyp-type peroxidases from Pseudomonas fluorescens Pf-5: Oxidation of Mn(II) and polymeric lignin by Dyp1B. Arch. Biochem. Biophys. 574 , 93–98 (2015).

Rhee, S. G. Overview on Peroxiredoxin. Mol. Cells 39 , 1–5 (2016).

Kameshwar, A. K. S. & Qin, W. Qualitative and Quantitative Methods for Isolation and Characterization of Lignin-Modifying Enzymes Secreted by Microorganisms. Bioenergy Res. 10 , 248–266 (2017).

Axelsson, L. et al . Perspective: Jatropha cultivation in southern India: Assessing farmers’ experiences. Biofuels, Bioprod. Biorefining 6 , 246–256 (2012).

Reiss, R. et al . Laccase versus Laccase-Like Multi-Copper Oxidase: A Comparative Study of Similar Enzymes with Diverse Substrate Spectra. PLoS One 8 , e65633 (2013).

Article   ADS   CAS   PubMed   PubMed Central   Google Scholar  

Granja-Travez, R. S. et al . Structural and functional characterisation of multi-copper oxidase CueO from lignin-degrading bacterium Ochrobactrum sp. reveal its activity towards lignin model compounds and lignosulfonate. FEBS J . 1–17, https://doi.org/10.1111/febs.14437 (2018).

Abdelaziz, O. Y. et al . Biological valorization of low molecular weight lignin. Biotechnol. Adv. 34 , 1318–1346 (2016).

Sato, Y. et al . Identification of three alcohol dehydrogenase genes involved in the stereospecific catabolism of arylglycerol-beta-aryl ether by Sphingobium sp. strain SYK-6. Appl. Environ. Microbiol. 75 , 5195–201 (2009).

Ichinose, H. Cytochrome P450 of wood-rotting basidiomycetes and biotechnological applications. Biotechnol. Appl. Biochem. 60 , 71–81 (2013).

Janusz, G. et al . Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution. FEMS Microbiol. Rev. 049 , 941–962 (2017).

Bugg, T. D. H., Ahmad, M., Hardiman, E. M. & Rahmanpour, R. Pathways for degradation of lignin in bacteria and fungi. Natural Product Reports 28 , 1883–1896 (2011).

Lee, S.-S., Moon, D.-S., Choi, H. T. & Song, H.-G. Purification and characterization of an intracellular NADH: quinone reductase from Trametes versicolor. J. Microbiol. 45 , 333–8 (2007).

CAS   PubMed   Google Scholar  

Kumar, M., Mishra, A., Singh, S. S., Srivastava, S. & Thakur, I. S. Expression and characterization of novel laccase gene from Pandoraea sp. ISTKB and its application. Int. J. Biol. Macromol. 115 , 308–316 (2018).

Wischgoll, S. et al . Gene clusters involved in anaerobic benzoate degradation of Geobacter metallireducens. Mol. Microbiol. 58 , 1238–1252 (2005).

Fuchs, G., Boll, M. & Heider, J. Microbial degradation of aromatic compounds- From one strategy to four. Nature Reviews Microbiology 9 , 803–816 (2011).

Kumar, M. et al . Genomic and proteomic analysis of lignin degrading and polyhydroxyalkanoate accumulating β-proteobacterium Pandoraea sp. ISTKB. Biotechnol. Biofuels 11 , 154 (2018).

Tahir, A. A. et al . Microbial diversity in decaying oil palm empty fruit bunches (OPEFB) and isolation of lignin-degrading bacteria from a tropical environment. Microbes Environ. 34 , 161–168 (2019).

Guillen, F., Martinez, A. T. & Jesus Martinez, M. Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii. Eur J Biochem. 209 , 603–611 (1992).

Asikkala, J., Tamminen, T. & Argyropoulos, D. S. Accurate and Reproducible Determination of Lignin Molar Mass by Acetobromination. J. Agric. Food Chem. 60 , 8968–8973 (2012).

Kumar, S., Stecher, G., Tamura, K. & Dudley, J. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets Downloaded from. Mol. Biol. Evol. 33 , 1870–1874 (2016).

Download references

Acknowledgements

The authors are grateful to Universiti Teknologi, Malaysia, for their financial support through a Research University Grant (GUP) Tier 1 (PY/2014/01793).

Author information

These authors contributed equally: Fatimah Azizah Riyadi and Analhuda Abdullah Tahir.

Authors and Affiliations

Department of Environmental Engineering and Green Technology, Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, Jalan Sultan Yahya Petra, 54100, Kuala Lumpur, Malaysia

Fatimah Azizah Riyadi, Analhuda Abdullah Tahir, Nurtasbiyah Yusof, Nurul Syazwani Ahmad Sabri, Megat Johari Megat Mohd Noor, Fazrena Nadia M. D. Akhir & Zuriati Zakaria

Department of Mechanical Precision Engineering, Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, Jalan Sultan Yahya Petra, 54100, Kuala Lumpur, Malaysia

Nor’azizi Othman

Department of Chemical Process Engineering, Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, Jalan Sultan Yahya Petra, 54100, Kuala Lumpur, Malaysia

Hirofumi Hara

You can also search for this author in PubMed   Google Scholar

Contributions

F.A.R. and A.A.T. contributed equally to the manuscript, and the experiments were mainly carried out by both. N.Y. and N.S.A.S. performed the draft genome sequencing by an Ion S5 XL sequencer. F.A.R., A.A.T., M.J.M.M.N., F.N.M.A., N.O., Z.Z. and H.H. contributed to writing, reading and approving the final manuscript.

Corresponding author

Correspondence to Hirofumi Hara .

Ethics declarations

Competing interests.

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary information., rights and permissions.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Cite this article.

Riyadi, F.A., Tahir, A.A., Yusof, N. et al. Enzymatic and genetic characterization of lignin depolymerization by Streptomyces sp. S6 isolated from a tropical environment. Sci Rep 10 , 7813 (2020). https://doi.org/10.1038/s41598-020-64817-4

Download citation

Received : 03 September 2019

Accepted : 23 April 2020

Published : 08 May 2020

DOI : https://doi.org/10.1038/s41598-020-64817-4

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

This article is cited by

Bacterial transformation of lignin: key enzymes and high-value products.

• Hongliang Huo

Biotechnology for Biofuels and Bioproducts (2024)

Palm oil decanter cake wastes as alternative nutrient sources for production of enzymes from Streptomyces philanthi RM-1-138 and the efficacy of its culture filtrate as an antimicrobial agent against plant pathogenic fungi and bacteria

• Sawai Boukaew
• Poonsuk Prasertsan
• Benjamas Cheirsilp

Biomass Conversion and Biorefinery (2024)

Sustainable bioethanol production from enzymatically hydrolyzed second-generation Posidonia oceanica waste using stable Microbacterium metallidurans carbohydrate-active enzymes as biocatalysts

• Afwa Gorrab
• Mohamed Neifar

Biomass Conversion and Biorefinery (2023)

Molecular Cloning and Characterization of Peroxidase from Haloferax volcanii

• Lakshmi Kasirajan
• Keerthana Kamaraj
• Julie A. Maupin-Furlow

Sugar Tech (2023)

Highly stable and tunable peptoid/hemin enzymatic mimetics with natural peroxidase-like activities

• Tengyue Jian
• Yicheng Zhou
• Chun-Long Chen

Nature Communications (2022)

By submitting a comment you agree to abide by our Terms and Community Guidelines . If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Quick links

• Explore articles by subject
• Guide to authors
• Editorial policies

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Lignin, from biomass waste to sustainable materials for food packaging, antiviral coatings and agriculture

Our planet has been challenged for years by our reckless use of fossil fuels, which has led to the ongoing climate change and energy crisis. The transition to the use of renewable alternative sources has now became urgent and inevitable. The valorization of lignin can be a key component of this transition, and this Thesis aims to contribute promoting this process. Lignin is an aromatic polymer that constitutes approximately one third of the total lignocellulosic biomass and is isolated in huge quantities as a waste material of biofuel and paper production. About 98% of the 100 million tons of lignin produced each year is simply burned as low-value fuel, so this renewable polymer is widely available at very low cost. Lignin has valuable properties that make it a promising material for numerous applications. Despite the recent interest in lignin valorization from industry and researchers, this natural polymer is far from being fully exploited due to several limitations. The overall scope of this PhD Thesis is to address some of the challenges in lignin employment and to find new applications for this underutilized material. Chapter 1 defines the main lignin features and describes how this polymer can be utilized as filler for food packaging films, as antimicrobial agent and as fertilizer. This chapter also points out the main challenges and limitations for the lignin involvement in these three fields of application. In Chapter 2, lignin nanoparticles have been functionalized and efficiently incorporated into a film of poly(lactic acid), a biodegradable polymer already utilized in sustainable food packaging. The lignin nanoparticles addition provided the final film with outstanding mechanical properties, antioxidant and UV-barrier activities, improving its performance as food packaging material. Chapter 3 explores the use of lignin as an antiviral coating. Viruses can transmit through the contact with contaminated surfaces and the systematic disinfection of surfaces is labor and time consuming, hence the development of coatings able to directly inactivate viruses would be useful to minimize the contamination risk. All the antiviral coatings known so far are inappropriate for the application on a large scale, while lignin would be an abundant and cheap solution. This chapter investigates the activity of lignin coatings against herpes simplex virus type 2 and examines the mechanism of viral inactivation through reactive oxygen species generation on the lignin surfaces. Chapter 4 describes the preparation of lignin nanoparticles bearing nitrogen and phosphorous, which are designed for the application as controlled-release fertilizers. These particles were conceived to be degraded by an enzyme produced by the plant roots, so that they can release the nutrients mainly in the plant proximity. The final purpose of the research work outlined in this chapter is to develop nano-fertilizers with efficient delivery of the active principle only where it is needed, reducing the waste of nutrients in the environment that characterizes the current agriculture practices. This Thesis work was accomplished hoping to contribute to the transition to a more careful and sustainable use of the resources our planet is providing us, starting with the valorization of an underutilized renewable biopolymer to address a wide spectrum of global challenges.

EPFL_TH10073.pdf

1a5480395a98acf6033c6d6235a10867

Synthesis, Characterization, and Applications of Polymer-grafted Lignin Surfactants

Degree type.

• Dissertation

Degree Name

• Doctor of Philosophy (PhD)

Usage metrics

• Chemical Sciences not elsewhere classified

Green Chemistry

A sustainable approach for lignin valorization by heterogeneous photocatalysis.

* Corresponding authors

a State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, P. R. China E-mail: [email protected]

b College of Chemistry, New Campus, Fuzhou University, Fuzhou, P. R. China

c Institute of Physical Chemistry of the Polish Academy of Sciences, ul. Kasprzaka 44/52, 01-224, Warsaw, Poland E-mail: [email protected]

The depletion of the Earth's fossil fuel reserves and the rapid increase in the emission of greenhouse gases and other environmental pollutants are driving the development of renewable energy technologies. Lignin is one of the three main subcomponents of lignocellulosic biomass in terrestrial ecosystems and makes up nearly 30% of the organic carbon sequestered in the biosphere. As a result of its rich content of aromatic carbon, lignin has the potential to be decomposed to yield valuable chemicals and alternatives to fossil fuels. However, the complex and stable chemical bonds of lignin make the depolymerization of lignin a difficult challenge with regard to its valorization. In this review, we highlight recent advances in the selective decomposition of lignin-based compounds via photocatalysis into other value-added chemicals and the treatment of waste water containing lignin. The photocatalytic transformation of lignin under mild conditions is particularly promising.

Article information

Download citation, permissions.

S. Li, S. Liu, J. C. Colmenares and Y. Xu, Green Chem. , 2016,  18 , 594 DOI: 10.1039/C5GC02109J

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page .

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page .

Read more about how to correctly acknowledge RSC content .

Social activity

Search articles by author, advertisements.

• CNRS - National Center for Scientific Research
• Posted on: 24 May 2024

PhD offer: Study of the pyrolysis of lignins extracted from black liquors (M/F)

Job Information

Offer description.

This thesis will take place at the University of Lorraine (UL) at the Reaction and Process Engineering Laboratory (LRGP – UMR CNRS 7274) in Nancy (54000, France).

The subject of this thesis concerns the precipitation step of lignin present in black liquors assisted by CO2 coupled with a conversion by pyrolysis to produce molecules of interest (phenols). One of the technological issue associated with the lignin pyrolysis is the agglomeration and clogging of the reactors. Indeed, during pyrolysis, the lignins form a sticky reactive intermediate material converted into char and liquid products (bio-oils) generated in the form of aerosols or vapors condensed in pyrolysis units. The scientific challenge of lignin pyrolysis is to control the composition of the liquids (monomers, suitable oligomers) and the structure of the char (biochar) produced. The objectives of this doctorate are: - ) to optimize the lignin precipitation step from black liquors from the DREAM project in a semi-continuous reactor: study of the effect of temperature, pressure, pH. Characterize the products obtained (GC, LC, NMR, IR, thermal analyses, etc.) -) study the pyrolysis of lignins obtained in a fluidized bed reactor (and others): flow rates, temperatures, fluidization behavior of the bed depending on the lignins. Hydrodynamic study by in-situ visualization of the reactor. Characterization of the products obtained (GC, LC, NMR, IR, thermal analyses, etc.).

The development of alternative biosourced processes is one of the major challenges of a sustainable economy concerned with preserving the environment. In pulp mills (from wood raw material to pulp), cellulose (fibers) are separated from lignin, hemicellulose and inorganic molecules. Around 100 million tonnes of wood are recovered for a production of 85 million tonnes of pulp in Europe (2022 data, including the use of recycled paper – 185 million tonnes worldwide, 7 million tonnes in France). The most used process is the Kraft process (90% of world production) which involves the treatment (digestion) of wood fibers in an aqueous solution called white liquor containing sodium hydroxide and sodium sulphide. Thus, black liquors (BL) are a waste stream from the Kraft process obtained after delignification of the biomass (wood) during the digestion operation. With more than 500 million tonnes/year of BL generated worldwide, the treatment and recovery of BL is an important industrial and environmental issue. Currently, BLs are concentrated by multiple evaporator systems and are then burned to regenerate inorganic materials and recover heat necessary for the Kraft process. Since evaporation systems have a fixed capacity, they currently constitute a bottleneck compared to a potential increase in production within factories using the Kraft process. In fact, these factories cannot produce more paper pulp without taking the risk of not being able to regenerate all of the by-products formed. Thus, the exploration of alternative processes for a portion of the BL produced could benefit pulp mills by reducing CO2 emissions and producing a wide variety of biosourced compounds: lignin, phenolic compounds, aromatics, acids. In the medium term, these pulp mills have the potential to transform into integrated forest biorefineries, producing a wide range of bio-sourced products. Numerous valorization strategies have been explored to extract/separate the compounds of interest (membrane filtration, liquid-liquid extraction, decantation, distillation) or to transform BL directly into energy (gasification, pyrolysis, hydrothermal treatment). It is in this context of need for development that the EIC Pathfinder DREAM project (processing complex matrices: Description, REAction-separation, Modelling) financed by the European Council takes place. The DREAM project aims to contribute to major scientific advances in the study of complex matrices (black liquor as a case study) with potentially future “on-site” industrial development. Several separation/extraction routes are studied in the DREAM project (membrane separation, precipitation, membrane filtration) followed by several transformation/purification routes (Reactive distillation, thermochemical processes).

Where to apply

Requirements, additional information.

https://cordis.europa.eu/project/id/101130523

Work Location(s)

Share this page.

• Corpus ID: 92329892

Production of vanillin from lignin present in the kraft black liquor of the pulp and paper industry

• José P. Araujo
• Published 2008

Figures and Tables from this paper

20 Citations

Kinetics of oxidative degradation of lignin-based phenolic compounds in batch reactor, isolation of phenolic monomers from kraft lignin using a magnetically recyclable tempo nanocatalyst, integrated process for vanillin and syringaldehyde production from kraft lignin, vanillin production from lignin and its use as a renewable chemical, lignin as source of fine chemicals: vanillin and syringaldehyde, production of vanillin from lignin: the relationship between β-o-4 linkages and vanillin yield, recovery of vanillin from kraft lignin depolymerization with water as desorption eluent, extraction, modification and characterization of lignin from oil palm fronds as corrosion inhibitors for mild steel in acidic solution.

• Highly Influenced

Added-Value Chemicals from Lignin Oxidation

Catalytic depolymerization of date palm waste to valuable c5–c12 compounds, 41 references, organic processes to pharmaceutical chemicals based on fine chemicals from lignosulfonates, kinetics of vanillin production from kraft lignin oxidation.

• Highly Influential

THE KRAFT PULP MILL AS A BIOREFINERY

Kinetics of vanillin oxidation, oxidation of vanillin by a new oxidant diperiodatoargentate(iii) in aqueous alkaline medium, chemistry and technology of agrochemical formulations, packed bed columns: for absorption, desorption, rectification and direct heat transfer, a method for calculating effective interfacial area of structured packed distillation columns under elevated pressures, residence time distribution and hold-up in a cocurrent upflow packed bed reactor at elevated pressure, numerical modeling of the drying, devolatilization and char conversion processes of black liquor droplets, related papers.

Showing 1 through 3 of 0 Related Papers

Geosciences Princeton University

Congratulations to dr. naomi intrator for successfully defending her ph.d. thesis.