Isolation and Characterization of Potential Lignin Peroxidase-Producing Bacteria from Compost Samples at Richards Bay (South Africa)

Lignocellulose is a significant plant component and varies among plant species (de Gonzalo et al. 2016). The demand for value-added products such as organic acids, biofuel, and microbial polysaccharides from lignocellulose biomass is increasing worldwide (Chaturvedi and Verma 2013). It is due to the renewable and abundant nature of the lignocellulose biomass. However, the recalcitrant nature of lignin remains a major bottleneck to unleashing useful products during various industrial operations. Due to inadequate treatment and waste management plans, lignin waste biomass from industrial and agricultural sectors continue to pose an environmental threat (Falade et al. 2017). Hence, valorizing lignocellulose waste is a welcome call for a proper waste management strategy.

Despite the commonly used chemical pre-treatment strategies to delignify lignocellulose biomass, there is a growing interest in biological delignification. In addition to the high capability of microorganisms to effectively degrade lignin, biological hydrolysis of lignin is also saddled with limited drawbacks (Datta et al. 2017). Unlike the chemical approach associated with high energy requirements and environmental threats, the biological hydrolysis of lignin is cost-effective, ecofriendly, and reduces environmental pollutants (Kumar and Chandra 2020).

The high capability of microorganisms to effectively degrade lignin is due to their ability to produce ligninmodifying enzymes (LMEs). Among the LMEs (laccase, manganese peroxidase, versatile peroxidase, etc.), lignin peroxidase is the most effective (Al-Dhabaan 2019). Lignin peroxidase (LiP), a heme peroxidase, utilizes hydrogen peroxide to oxidize a wide variety of aromatic model compounds (of a phenolic and non-phenolic nature) of lignin into mono-aromatic structures (Janusz et al. 2017). LiP thus could play a pivotal role in treating industrial waste and degradation of xenobiotics such as dyes, organophosphorus, and polycyclic aromatic hydrocarbons (Falade et al. 2017). Such versatile industrial applications of LiP increase the need to search for LiP-producing microorganisms.

Although white rot basidiomycetes have intensively been studied as a source of potent ligninolytic extracellular oxidative enzymes, the ligninolytic use of fungus is limited (Su et al. 2018). Bacteria candidature as ligninolytic agents is currently gaining attention in research. It is due to lesser ethical issues regarding bacteria use, environmental adaptation, spontaneous growth, and the ease of genetic manipulation of bacteria (Tian 2016). Hence, there is a growing interest in exploring bacteria as potential ligninolytic agents. In searching for bacteria that can produce lignin-modifying enzymes (LMEs), bacterial species classified into actinomycetes, α-Proteobacteria, and γ-Proteobacteria have been identified (Wang et al. 2016). Such bacteria include Streptomyces viridosporus, and strains of Nocardia and Pseudomonas (Zimmermann 1990; Bugg et al. 2011).

The Richards Bay environment, a central industrial hub in the Kwa-Zulu Natal province of South Africa, is characterized by loops of hemp of lignin-based wastes and untreated industrial wastewater, posing environmental hazards (Okello et al. 2022). Such an environment might serve as a reservoir for bacterial genome pool to exploit novel bacteria strains with excellent lignin-modifying enzymes. Therefore, this study intended to isolate and characterize potential ligninolytic bacterial strains with lignin peroxidase production capacity from compost samples: sawdust, decaying soil, and cow dung from the Richards Bay environment.

This study isolated twenty-six potential ligninolytic bacteria from samples collected in the Richards Bay environment. Most of the isolates were identified (Table SI) as P. aeruginosa (88%), Enterobacter spp. (8%), and E. coli (4%). These ligninolytic bacteria were all found to be γ-Proteobacteria. The phylogenetic tree, showing the relationship between the P. aeruginosa and the Enterobacter hormaechei in this study, and other ligninolytic bacteria is presented in Fig. 3. The identified isolates all showed 99% similarity to the previously isolated strains (Xiong et al. 2020).

Fig. 3.

According to Lopez et al. (2006), using lignin as a carbon source for the selection of ligninolytic microorganisms is regarded as one of the most appropriate methods worldwide. However, the utilization of lignin model compounds such as veratryl and guaiacol alcohols, and the decolorization of industrial dyes as exhibited by the isolates (Table I and II), is the evidence that the colonies have the potential to utilize and degrade lignin (Husain 2006; Bandounas et al. 2011; Falade et al. 2017).

However, growth on polymeric lignin or lignin monomers is not necessarily a good measure of the ligninolytic potential. The ligninolytic potential, independent from lignin utilization, was studied with the decolorization of synthetic lignin-like dyes (Falade et al. 2017). The selected organisms were able to decolorize Azure B. Although most LMEs can decolorize various dyes, previous studies (Falade et al. 2017) have revealed that Azure B decolorization is associated explicitly with high redox potential agents like lignin peroxidases (LiP). Other LMEs, such as laccase and manganese peroxidase-producing bacteria, cannot decolorize Azure B (AZ). Since fifteen of the isolated ligninolytic bacteria degraded AZ (Table II), it is apparent that these bacterial isolates were potential LiP producers. However, out of the 15 isolates, 10 isolates were able to decolorize Azure B only. These pure colonies gave a positive pyrogallol test for peroxidase, and the supernatant of cultures quantitatively revealed the activity of peroxidase (Arora and Gill 2011). Therefore, they were selected as potential lignin peroxidase producers, and this was confirmed by the production of a crude enzyme complex that contained a peroxidase, most likely, a lignin peroxidase. They were not laccase producers (Table III). All the selected isolates also showed high catalase and oxidase activities and were capable of hydrolyzing starch (Table IV).

The lignin degradation capabilities of these bacterial strains were further examined, and the evidence for ligninolytic activity was based on) the bacterial growth on lignin enriched medium and the lignin degradation. Most of the tested bacterial strains were able to degrade lignin. The isolated organisms were cultured on lignin (with 1% glucose), and after a period of cultivation, phenolic (or derived lignin monomers) appeared and increased with growth in the culture. We believe that the organisms produced these phenolic compounds in the culture as a result of lignin (a polyphenolic complex) being degraded. We also agree that the lignin degradation should be monitored as directly as possible. However, synthetic dyes may mimic lignin (Sana et al. 2017). In this present study, the lignin degradation was prominent with E. coli LR0250096.1 and P. aeruginosa CP031449.2, as their growth increased with the degradation of lignin (Fig. 1). These results are consistent with literature reports on the ligninolytic activity of Pseudomonas sp. and E. coli species (Bandounas et al. 2011; Sana et al. 2017).

Interestingly, of 26 isolates obtained, ten were identified as true lignin peroxidase producers with E. coli LR0250096.1, and P. aeruginosa CP031449.2 considered the best lignin peroxidase producers, exhibiting activity of about 1.99 × 10^–1 U/l (Table VI, Fig. 2). Other bacteria strains such as Klebsiella pneumonia, Bacillus species, Citrobacter species, Serratia marcescens, and Pseudomanos spp. have also been reported to have LMEs-producing potential (Zainith et al. 2019; Kumar and Chandra 2020).

This study attempted to isolate and characterize lignin-degrading bacteria capable of producing lignin peroxidase. The isolated Pseudomonas and Enterobacter species and E. coli were identified as true lignin peroxidase producers. The highest ligninolytic activity exhibited by P. aeruginosa (CP031449.2) and E. coli (LR025096.1) suggests that these isolates could be exploited in the industrial treatment of lignin and detoxification of recalcitrant industrial dyes that invariably end up in the environment. For further studies, enzyme purification, enzyme’s efficiency, and the mechanism of its action will be ascertained.