Three microbial soil inhabitants identified as Pandoraea norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003 were isolated as potential lignin depolymerizing bacteria. The isolated strains showed growth on both high and low-molecular weight lignin fractions, although growth of Bacillus sp. LD003 was relatively poor. Typical lignin-associated monomers were utilized to a rather limited extent by all three isolates. Remarkably, the isolated strains appeared to lack the ability to oxidize aromatic alcohols or aldehydes to their corresponding carboxylic acid form.
The ligninolytic potential of the isolates was furthermore assessed by establishing their ability to decolourize synthetic, lignin-like dyes. The recalcitrant thiazine dye Azure B (AB) is particularly suited for this purpose. This dye is decolourized by high redox potential agents, specifically LiP's [17, 40, 41], whereas it cannot be oxidized by nonperoxidase alcohol oxidases, MnP's or laccases alone [40, 42]. In contrast to the other two isolates, Bacillus sp. LD003 readily decolourized AB as well as most of the other lignin-mimicking dyes tested. Also other Bacillus species as well as members of the Streptomyces genus have been reported to degrade AB within 4 - 6 days. These bacteria were isolated from wooden objects, and decolourization of AB was measured to demonstrate lignin peroxidase activity . AB closely resembles methylene blue (MB) and toluidine blue O that were also readily degraded by Bacillus sp. LD003. MB has previously been found to be oxidized by lignin peroxidase [44, 45].
The seemingly contradictory finding that the highest ligninolytic potential appeared to be associated with the strain that showed poorest growth on lignin may be understood from an ecological perspective. Often, recalcitrant compounds such as lignin are degraded by microbial consortia in which the individual strains have specialized roles: some attack the complex substrate whereas others provide essential nutrients . Ligninolytic bacterial consortia can be found, e.g., in the gut of wood-feeding termites. Bacteria like Rhodococcus erythropolis, Burkholderia sp., Citrobacter sp. and Pseudomonas sp. have been isolated from the guts of wood-feeding termites and beetles. These bacteria typically degrade aromatic compounds [25, 48, 49], which suggests that they feed on the aromatic compounds liberated by the lignin degrading species of the gut microflora. However, lignin-degrading activity has also been reported for certain aromatic compound degraders such as Pseudomonas sp. and Burkholderia sp. Furthermore, genera such as Burkholderia, Pseudomonas, Sphingomonas, Bacillus and Pandoraea have been reported to degrade the structurally crucial biphenyl component of lignin, which composes up to 10% of the structure, depending on the lignin type [27, 50, 51].
Like in other lignin preparations, trace amounts of (hemi)-cellulose may be present in Kraft lignin. This however, is not likely to account for the observed growth on lignin, although the cellulolytic capacity of the isolated strains has not been investigated in detail. Many if not most soil bacteria have incomplete cellulolytic systems . Especially Pandoraea norimbergensis is unlikely to utilize cellulose, since it was unable to utilize glucose or cellobiose, both comprising cellulose . Indeed, several Bacillus sp. are able to utilize cellulose . The limited growth observed however, on both the high and low molecular weight lignin fractions, in combination with the ability to utilize certain lignin-model dyes clearly indicate the ligninolytic potential of this strain. Other Bacillus sp. have accordingly been reported to degrade Kraft lignin [55–57]. In addition, several Pseudomonas sp. are able to degrade various lignin preparations such as milled wood lignin, dioxane lignin and lignin from poplar wood , further supporting our findings.
In a ligninolytic consortium, Bacillus sp. LD003 may fulfill the role of lignin degrader that has to rely on other microbes for specific nutrients, as suggested by its requirement for yeast extract. The other isolates in this study, Pseudomonas sp. LD002 and P. norimbergensis LD001, showed lesser ligninolytic capacities, but utilized a somewhat wider range of aromatics and did not depend on additional nutrients. Thus, such strains may fulfill the role of nutrient-provider.
The bacterial isolates in this study appear to have an alternative type of ligninolytic system. The enzymes are presumably cell-surface associated, in view of the large size of lignin, whereas fungal lignin degradation occurs via extracellular enzymes and secreted secondary metabolites [59–62]. Thus, a new and presumably vast source may be tapped for novel ligninolytic enzyme activities. A few considerations, however, must be taken into account when hunting for novel ligninolytic activities for lignin valorization. First, the type of lignin to be valorized is a key factor, since the process by which it is obtained will result in structural modifications [15, 63, 64]. Thus, "natural" ligninolytic systems like those associated with white-rot fungi may not be the most efficient to valorize "industrial" lignin streams such as the Kraft lignin employed in this study. Furthermore, the most efficient lignin mineralizing strains may not be the most efficient lignin depolymerizers. Therefore, lignin degradation should be monitored as directly as possible. Ideally, the actual substrate should be used in degradation assays, but the heterogeneous nature of lignin severely complicates the analytics. Alternatively, synthetic dyes may be used to mimic lignin as we did in the present study. However, the ligninolytic activities obtained by this approach should be evaluated for their utility on the proper type of lignin.