Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

Abstract

Background: To expand on the range of products which can be obtained from lignocellulosic biomass, the lignin component should be utilized as feedstock for value-added chemicals such as substituted aromatics, instead of being incinerated for heat and energy. Enzymes could provide an effective means for lignin depolymerization into products of interest. In this study, soil bacteria were isolated by enrichment on Kraft lignin and evaluated for their ligninolytic potential as a source of novel enzymes for waste lignin valorization.

Results: Based on 16S rRNA gene sequencing and phenotypic characterization, the organisms were identified as Pandoraea norimbergensis LD001, Pseudomonas sp LD002 and Bacillus sp LD003. The ligninolytic capability of each of these isolates was assessed by growth on high-molecular weight and low-molecular weight lignin fractions, utilization of lignin-associated aromatic monomers and degradation of ligninolytic indicator dyes. Pandoraea norimbergensis LD001 and Pseudomonas sp. LD002 exhibited best growth on lignin fractions, but limited dye-decolourizing capacity. Bacillus sp. LD003, however, showed least efficient growth on lignin fractions but extensive dye-decolourizing capacity, with a particular preference for the recalcitrant phenothiazine dye class (Azure B, Methylene Blue and Toluidene Blue O).

Conclusions: Bacillus sp. LD003 was selected as a promising source of novel types of ligninolytic enzymes. Our observations suggested that lignin mineralization and depolymerization are separate events which place additional challenges on the screening of ligninolytic microorganisms for specific ligninolytic enzymes.

Figure 1

Growth of bacterial isolates on lignin. Growth of P. norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003 on a) HMW lignin fraction and b) LMW lignin fraction. Experiments were performed in 4-fold; mean values of CFUs are shown with error bars indicating the maximum deviation from the mean. * Last CFU count for Bacillus sp. LD003 was performed at 96 h instead of 120 h.

Figure 2

Decolourization of ligninolytic indicator dyes. Decolourization (% of initial value) of ligninolytic indicator dyes in LB medium, 25 h after dye addition to exponentially growing cultures of P. norimbergensis LD001, Pseudomonas sp. LD002 and Bacillus sp. LD003. Error bars indicate the maximum deviation from the mean of duplicate experiments. * indicates that dye adsorption to the cell pellet was observed after centrifugation. Dyes: Azure B (AB), Methylene blue (MB), Toluidene Blue O (TB), Malachite Green (MG), Congo red (CR), Xylidine ponceau (XP), Indigo Carmine (IC) and Remazol Brilliant Blue R (RBBR).

Figure 3

Decolourization zones in dye-containing plates. Decolourization zones in dye-containing plates after 72 h of incubation. a) LB with 25 mg/L Methylene Blue (MB); b) LB with 25 mg/L Azure B (AB); c) LB with 25 mg/L Toluidine Blue O (TB); d). MM + 20 mM citrate + 0,5 g/L YE with 25 mg/L Methylene Blue; e) MM + 20 mM citrate + 0,5 g/L YE with 25 mg/L Azure B; f) MM + 20 mM citrate + 0,5 g/L YE with 25 mg/L Toluidine Blue O; 1 - P. norimbergensis LD001, 2 - Pseudomonas sp. LD002, 3 - Bacillus sp. LD003. Experiment performed in duplicate.