Assessing Lignin Types To Screen Novel Biomass-Degrading

Letter pubs.acs.org/journal/ascecg

Assessing Lignin Types To Screen Novel Biomass-Degrading Microbial Strains: Synthetic Lignin as Useful Carbon Source Pere Picart,† Lotte Wiermans,‡ María Pérez-Sánchez,‡ Philipp M. Grande,‡ Anett Schallmey,*,† and Pablo Domínguez de María*,‡,§ †

Junior Professorship for Biocatalysis, Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany ‡ Institut für Technische und Makromolekulare Chemie (ITMC), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany § Sustainable Momentum, SL. Ap. Correos 3517, 35004 Las Palmas de Gran Canaria, Canary Islands, Spain S Supporting Information *

ABSTRACT: The isolation of lignin-degrading microbial strains may lead to the discovery of novel biocatalysts peroxidases, laccases, and β-etherasespotentially useful for lignin valorization. The inherent heterogeneity of lignin, together with the sometimes difficult accessibility to representative amounts of it, may become a hurdle for using lignin as a carbon source for screening purposes. This communication compares the screening of soil samples for lignin-degrading bacteria using as carbon sources either OrganoCat lignin or synthetically produced lignin. In both cases, the same microbial strains were isolated, suggesting that synthetic ligninstraightforwardly produced using peroxidases at laboratory scalecan be a valuable lignin substitute for microbial screenings and available in sufficient quantities. Likewise, OrganoCat lignin was dearomatized (50% and 100%) with a novel protocol using hydrogen peroxide and dimethylcarbonate, and the obtained derivatives were applied as carbon sources as well. In these cases, different microorganisms from those observed with real lignin derivatives were isolated from the same soil sample. Isolated microorganisms growing on nondegraded lignin polymers (OrganoCat and synthetic carbon sources) predominantly produced peroxidases, whereas strains growing on fully dearomatized lignins also secreted laccases. KEYWORDS: Lignin, Microorganisms, Synthetic lignin, Enzymes, Biorefineries

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ignin represents an under-exploited but potentially valuable renewable feedstock for its further use in biorefineries to deliver novel biofuels and biocommodities.1−4 Accounting for a substantial amount of lignocellulosic biomass (typically 20%−40%), the valorization of lignin is having presently an increasing interest, with considerable efforts at the academic level to establish novel (bio)catalytic strategies.5,6 Several enzymes, namely, peroxidases, laccases, and β-etherases, have been described to depolymerize lignin and have been biocatalytically characterized.7,8 Given the potential that lignin-degrading enzymes may have for its future implementation in biorefineries, the quest for novel biocatalysts with high robustness and outstanding catalytic performances is of utmost importance. In this respect, a common approach is the accomplishment of enrichment cultures with lignin as the sole carbon source and subsequent isolation of single microbial strains that are able to grow on lignin (Figure 1). In this line, several articles in the literature report on soil bacteria able to metabolize lignin,9−11 which were isolated from industrial lignin residues from the Kraft process (predominant © XXXX American Chemical Society

Figure 1. Common strategy to identify novel microorganisms with lignin-degrading capabilities.

Received: August 28, 2015 Revised: January 23, 2016

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DOI: 10.1021/acssuschemeng.5b00961 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Sustainable Chemistry & Engineering

(for presence of laccases, peroxidases and β-etherases) of the isolated strains were carried out. In the first set of experiments, OrganoCat lignin from beech wood and a synthetic lignin based on coniferyl alcohol polymerized in vitro with horseradish peroxidasewere used as carbon sources and obtained results compared. Quite remarkably, the same individual bacterial isolates were obtained as identified by partial 16S rRNA gene sequencing. The isolated strains were determined as Stenotrophomonas maltophilia, Shigella sonnei, Enterobacter sp., Pseudomonas sp., and Ralstonia sp. Moreover, in both cases, similar growthmeasured as colony-forming units, CFU mL−1was observed after 48 or 96 h of incubation (Figure 2), supporting the idea that synthetic

process in the pulping industry). In general, access to sufficient representative amounts of standardized lignin may be cumbersome, complicating the setup of reliable screening methods, which would need to be based on real lignin derivatives. Likewise, studies comparing the influence of lignin type (as carbon source) for identification of microbial strains present in the same soil sample have never been conducted. In this respect, the use of “synthetic lignin”, namely, dehydrogenative polymers, DHP, straightforwardly produced at laboratory scale using peroxidases,12 may represent a promising approach, provided that screening results would be comparable to those of natural lignin (that is, same lignin-isolating strains are obtained). Actually, synthetic lignin can be representatively produced in a simple and homogeneous way at sufficient amounts for (larger) fermentative purposes. Given the potential importance that this may have for future screenings of lignindegrading microbial strains, this communication compares natural and synthetic lignin as carbon sources for the first time. In order to perform enrichment cultures, a soil sample containing degraded wood was used as inoculum. Additionally, four different types of lignin were selected as the carbon source: (a) beech wood lignin obtained by the OrganoCat pretreatment process,13 (b) synthetic lignin (dehydrogenated polymer, DHP) produced in the laboratory using peroxidases,12 (c) two further OrganoCat beech wood lignins, in which a controlled dearomatization procedure (using diluted hydrogen peroxide and dimethylcarbonate, according to our recently reported protocol 14 ) was conducted to reach 50% and 100% dearomatization levels. The analytic characterization of these lignin samples has been published elsewhere (including 2DNMR, elemental analysis, FTIR, ESI-MS, etc.).12,14 Thus, the OrganoCat lignin shows the classic aromatic stretching bands at 1400−1600 cm−1 in FTIR, and ESI-MS analysis of this lignin reveals mainly two fragments of 1017.7 and 1142.2 g mol−1.14 Likewise, dearomatized lignin samples (50% and 100%) clearly show a decrease in the aromatic bands in the NMR analysis,12,14 with less or total absence of aromatic stretching bands in FTIR. This is combined with a considerable increase in the carbonylic groups band at ∼1600 cm−1, presumably related to carboxylic acids as well as a significant enhancement in oxygen content according to elemental composition studies (as expected being the dearomatization an oxidative process using hydrogen peroxide).14 Furthermore, ESI-MS studies reveal that those dearomatized samples are a complex mixture of smaller fragments, with a distribution pattern mostly ranging from 200 to 900 g mol−1.14 Actually, the visual appearance of the derivatives already suggests the production of smaller fragments, ranging from solid brown to black lignins (OrganoCat) to rather viscous yellow oil at 100% dearomatization.14 Finally, the ESI-MS of synthetic lignin showed a slightly smaller fragment distribution frame compared to OrganoCat lignin, ranging from 400 to 1000 g mol−1. The presence of the key bonds in lignin (e.g., β-O-4, β−β, etc.) was confirmed by NMR studies.12,14 For the microbial screenings, lignin-degrading microorganisms were enriched in liquid mineral medium (MM) with the above-described different types of lignin as the sole carbon source and using a suspension of the soil sample as inoculum (see the Experimental section, Supporting Information). After five successive transfers to fresh lignin-containing MM, individual colonies were obtained on Luria broth (LB) agar plates, and characterizations as well as enzymatic assessments

Figure 2. Growth of different bacterial isolates on natural (OrganoCat) and synthetic lignin.

lignin may be an excellent substitute for real lignin samples as the carbon source in microbial screenings. With regard to the identified microorganisms, the ability of degrading Kraft or alkali lignins has already been reported for Stenotrophomonas sp.,15 Enterobacter sp.,16 and Ralstonia sp. (syn. Cupriavidus),17 while genomic annotation of genes involved in the degradation of aromatic compounds and/or lignin has been recently described for Shigella sonnei18 and Pseudomonas sp.10,19 Subsequently, the dearomatized lignin samples (50% and 100% dearomatized) were used as the sole carbon source for enrichments based on the same soil sample. Given the oxidative dearomatization process of the ligninwith an increase in oxygen and carbonylic groups, as well as a reduction of pH in aqueous suspensions12,14it may be assumed that a higher number of carboxylic acids are present, together with shorter polymer fragments and less aromatic groups, as confirmed by NMR and ESI-MS,12,14 leading to more flexible lignin fibers.14 Due to the higher presence of carboxylic acids within the resulting modified lignin, the pH of the solution dropped to 4 when enrichment cultures were prepared (consistent with previous literature12). Under these more acidic conditions, two different species were isolated: Propionibacterium sp. and Exiguobacterium sp. (Figure 3), which are different from the five strains isolated on natural or synthetic lignin. A Propionibacterium strain has been previously reported to be able to degrade various aromatic hydrocarbons.20 Moreover, an B


Letter


peroxidases (MnP), and β-etherases was screened, using cellpellets and cell-free culture supernatants of isolated strains. βEtherases catalyze the cleavage of the β-O-4-arylether bonds, the most abundant ones present in lignin.22−24 Laccases oxidize the phenolic units in lignin and are also able to catalyze interunit bond cleavage (alkyl-aryl and Cα-Cβ cleavage) in lignin substrates in the presence of a mediator system. Lignin peroxidases are able to oxidize and cleave the recalcitrant nonphenolic structures that comprise the bulk of lignin, via Cα−Cβ rupture of the alkyl side chain. Also, manganese peroxidases oxidize Mn2+ to Mn3+, which can oxidize only the minor phenolic units in lignin. Overall results are summarized in Table 1. As a result, the five strains isolated on OrganoCat and synthetic lignin and the five strains isolated on dearomatized lignin displayed peroxidase activity, which seems to be necessary to metabolize any lignin-based polymer fragment. Such activity has also been found in other lignin-degrading microorganisms.25−27 The ligninolytic activity was furthermore assessed by investigating the ability of the microorganisms to decolourize synthetic lignin-like dyes in solid media. Azure B and methylene blue are especially suitable for this purpose because they can only be decolourized by high redox potential agents, specifically by lignin peroxidases,28,29 whereas they cannot be oxidized by nonperoxidase alcohol oxidases, manganese peroxidases, or laccases alone.28,30 Hence, all isolated strains except Ralstonia sp. showed decolourization zones in dye-containing plates after 48 h of growth (Figure 5). However, crude culture supernatants of the different isolated strains, using synthetic lignin or OrganoCat lignin to induce the respective degrading enzymes, did not exhibit any peroxidase activities, suggesting that the respective peroxidases might be cell-associated. This observation of peroxidases acting as cellbound catalysts has been noted in the open literature, suggesting that this phenomenon might be somehow abundant in nature.10 In contrast, laccase activity was only found in microorganisms growing on 100% dearomatized lignins, both at pH 4 and 7. This could be either due to differential induction of enzyme activities by polymeric and 100% dearomatized lignins or specific to the obtained microorganisms that were isolated on polymeric or dearomatized lignins. Interestingly, laccase activities were found both in the crude culture supernatant and the cell pellet, indicating the simultaneous presence of extracellular and intracellular laccases, consistent with data previously reported for Bacillus pumilus and Bacillus atrophaeus.11 It has been suggested that the different location of laccases could be related to their physiological functions,31 and in vitro laccases may actually catalyze polymerizations as well as depolymerizations (with mediators).32 From our observed results, it seems that peroxidases might be crucial to degrade lignin polymers, while laccases would be beneficial but not totally necessary for such purposes. In subsequent research steps, these novel potential ligninolytic enzymes should be identified, cloned, and heterologously expressed to purify them and to assess in vitro which lignin bonds are actually being degraded.

Figure 3. Growth of different bacterial isolates at pH 4 using dearomatized lignins (50% and 100% dearomatized samples) as sole carbon source.

Exiguobacterium sp. isolated from dyestuff-contaminated soil has been shown to possess lignin peroxidase and laccase activities,21 similar to the Exiguobacterium isolate reported herein (see below). Subsequently, the microbial screenings with dearomatized lignins (50% and 100%) were repeated, yet in this case adjusting pH to 7 after addition of the carbon source. This time, three different bacterial strains were obtained: Shigella sp., Enterobacter ludwigii, and Delftia sp. Again, for both dearomatized lignins, the same three species were isolated; yet at 100% dearomatization, less growth was observed than for the 50% dearomatized lignin as carbon source (Figure 4). Once all the different strains were isolated, the ligninolytic potential of these microorganisms was assessed. Specifically, the presence of laccases, lignin peroxidases (LiP), manganese

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CONCLUSIONS A comparison between different lignins and related derivatives as carbon sources to screen soil samples to identify microbial strains has been performed. Studies using OrganoCat lignin or synthetic lignin (with the same soil sample) yielded the same

Figure 4. Growth of microbial isolates using dearomatized lignins (50% and 100%) as carbon source at pH 7. C


Letter

ACS Sustainable Chemistry & Engineering Table 1. Screening of Isolated Lignin-Degrading Strains for Different Ligninolytic Enzymatic Activities laccase

lignin peroxidase

manganese peroxidase

β-etherase

strain

lignin

sup.a

cellb

sup.a

cellb

sup.a

cellb

sup.a

cellb

Ralstonia sp.c

OrganoCat synthetic OrganoCat synthetic OrganoCat synthetic OrganoCat synthetic OrganoCat synthetic dearo. 50% dearo. 100% dearo. 50% dearo. 100% dearo. 50% dearo. 100% dearo. 50% dearo. 100% dearo. 50% dearo. 100%

nd

nd

nd

nd

nd

+

nd

nd

nd

nd

nd

+

nd

nd

nd

nd

nd

nd

nd

+

nd

nd

nd

nd

nd

nd

nd

+

nd

nd

nd

nd

nd

nd

nd

++

nd

nd

nd

nd

nd + nd + nd + nd + nd +

nd ++ nd ++ nd ++ nd + nd ++

nd nd nd nd nd nd nd nd nd nd

++ ++ ++ ++ +++ +++ + + + +





Pseudomonas sp.c Enterobacter sp.c Shigella sonneic Stenotrophomonas maltophiliac Shigella sp.d Enterobacter ludwigiid Delftia sp.d Propionibacterium sp.e Exiguobacterium sp.e

a Activity in the supernatant after removal of cells by centrifugation. bActivity in cell pellets and cell-free extracts. cSame results were obtained using either natural (OrganoCat) or synthetic lignin. dStrains isolated with dearomatized lignin as carbon source at pH 7. eStrains isolated with dearomatized lignin as carbon source at pH 4. nd: not detected.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b00961. Materials and methods (S2). Experimental production of OrganoCat lignin (S2). Production of synthetic lignin (S2). Protocol for catalyst-free lignin dearomatization (S3). Protocol for isolation and identification of lignindegrading bacteria (S3). Protocol for enzymatic activities (S4). Dye decolorization assays (S5). References (S6). (PDF)

Figure 5. Agar plate-based colorimetric activity assays to monitor lignin peroxidase activity.

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isolated microorganisms. Conversely, when dearomatized lignins (50% or 100% dearomatization) were assessed, different microorganisms were isolated, either at pH 4 or 7. When OrganoCat lignin and synthetic lignin are used as carbon sources, the presence of peroxidases seemed to be crucial for the microorganisms, suggesting that peroxidases are mandatory for lignin-degrading microbial strains. Laccases appeared only in isolated microorganisms when dearomatized lignin samples were employed. Overall results suggest that synthetic lignin may become a useful tool for screening of novel microorganisms, as it can be straightforwardly produced at laboratory scale and leads to the same isolated strains than those obtained for real lignin samples. Since lignin samples may be not accessible in large scale and in homogeneous (consistent) form, the delivery of synthetic carbon sources may become a very useful tool. On the basis of this proof-of-concept, more research is needed to optimize the growth conditions of the isolated microorganisms, as well as to characterize the new enzymes that might be useful for future in vitro lignin valorization processes.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +49 531 391 5803 (A.S.). *E-mail: [email protected]. Tel.: +34 609 565237 (P.D.d.M.). Present Address

A. Schallmey: Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was performed as part of the Cluster of Excellence “Tailor-Made Fuels from Biomass”, which is funded by the Excellence Initiative of the German Research Foundation to promote science and research at German universities. D


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O-aryl alkyl ethers and various aromatic hydrocarbons. Chemosphere 2009, 75, 1287−1293. (21) Dhanve, R. S.; Shedbalkar, U. U.; Jadhav, J. P. Biodegradation of diazo reactive dye Navy blue HE2R (Reactive blue 172) by an isolated Exiguobacterium sp. RD3. Biotechnol. Bioprocess Eng. 2008, 13, 53−60. (22) Picart, P.; Müller, C.; Mottweiler, J.; Bolm, C.; Domínguez de María, P.; Schallmey, A.; Wiermans, L. From gene towards selective biomass valorization: Novel bacterial β-etheraes with activity on ligninlike polymers. ChemSusChem 2014, 7, 3164−3171. (23) Picart, P.; Sevenich, M.; Domínguez de María, P.; Schallmey, A. Exploring gluthatione lyases as biocatalysts: paving the way for enzymatic lignin depolymerization and future stereoselective applications. Green Chem. 2015, 17, 4931−4940. (24) Picart, P.; Domínguez de María, P.; Schallmey, A. From gene to biorefinery: Microbial β-etherases as promising biocatalysts for lignin valorization. Front. Microbiol. 2015, 6, 916. (25) Roberts, J. N.; Singh, R.; Grigg, J. C.; Murphy, M. E.; Bugg, T. D. H.; Eltis, L. D. Characterization of dye-decolorizing peroxidases from Rhodococcus jostii RHA1. Biochemistry 2011, 50, 5108−5119. (26) Ahmad, M.; Roberts, J. N.; Hardiman, E. M.; Singh, R.; Eltis, L. D.; Bugg, T. D. H. Identification of DypB from Rhodococcus jostii RHA1 as a lignin peroxidase. Biochemistry 2011, 50, 5096−5107. (27) Singh, R.; Grigg, J. C.; Qin, W.; Kadla, J. F.; Murphy, M. E.; Eltis, L. D. Improved manganese-oxidizing activity of DypB, a peroxidase from a lignolytic bacterium. ACS Chem. Biol. 2013, 8, 700−706. (28) Archibald, F. S. A new assay for lignin-type peroxidases employing the dye azure B. Appl. Environ. Microbiol. 1992, 58, 3110− 3116. (29) Aguiar, A.; Ferraz, A. Fe3+ - and Cu2+ - reduction by phenol derivatives associated with Azure B degradation in Fenton-like reactions. Chemosphere 2007, 66, 947−954. (30) Arora, D. S.; Gill, P. K. Comparison of two assay procedures for lignin peroxidase. Enzyme Microb. Technol. 2001, 28, 602−605. (31) Santhanam, N.; Vivanco, J. M.; Decker, S. R.; Reardon, F. Expression of industrially relevant laccases: prokaryotic style. Trends Biotechnol. 2011, 29, 480−489. (32) Munk, L.; Sitarz, A. K.; Kalyani, D. C.; Mikkelsen, J. D.; Meyer, A. S. Can laccases catalyze bond cleavage in lignin? Biotechnol. Adv. 2015, 33, 13−24.

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Assessing Lignin Types To Screen Novel Biomass-Degrading

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