Biological conversion of aromatic monolignol compounds by a Pseudomonas isolate from sediments of the Baltic Sea

Lignin is one of the most plentiful biopolymers on Earth. It is a complex alkyl–aromatic heteropolymer found in the plant cell wall, which provides strength and protection to terrestrial plants. Currently, technical lignin is produced from either the pulp/paper industries and/or cellulosic ethanol biorefineries, where it is mainly used as an energy source to produce steam and electricity. Lignin is underexploited as a chemical feedstock because of its heterogeneity and intractable structure (Ayyachamy et al. 2013). The valorization of lignin to produce renewable fuels and chemicals is important to further develop the biorefineries (Beckham et al. 2016; Camarero et al. 2014; Rodriguez et al. 2017). To allow lignin to be used as a substrate for bioconversion, a depolymerization step is essential to generate a combination of monomeric, dimeric and oligomeric lignin-based compounds (Rinaldi et al. 2016; Xu et al. 2014). These can be further catabolized into various bio-based compounds of economic value for lignocellulosic biorefineries (Abdelaziz et al. 2016; Salvachúa et al. 2015).

Lignin in nature is degraded mainly by extracellular peroxidases and laccases, secreted by white-rot and brown-rot fungi. The action of these enzymes on lignin results in a diverse range of low molecular weight aromatic fragments (Martínez et al. 2005). Due to the widespread availability of these aromatic molecules in the environment, several microbes have developed catabolic pathways for these (Makela et al. 2015). There have been many studies on a variety of fungal species, which can utilize lignin-related compounds with the help of redox mediators (Martínez et al. 2005). However, their commercial application is limited by slow growth rates and difficulties related to fungal genetic manipulation (Bugg et al. 2011a).

In addition to fungi, there are several bacterial species capable of catabolizing lignin or lignin-related aromatic compounds (Brown and Chang 2014; Bugg et al. 2011b). Bacteria have certain benefits over fungi, e.g. easy genetic engineering, fast growth and ease of cultivation. Reported bacteria with ability to convert aromatics include Pseudomonas putida KT2440 (Jiménez et al. 2002; Ravi et al. 2017), Cupriavidus necator JMP134 (Pérez-Pantoja et al. 2008), Rhodococcus opacus (Zhao et al. 2016), Rhodococcus jostii RHA1 (Ahmad et al. 2011), Acinetobacter baylyi ADP1 (Barbe et al. 2004), Amycolatopsis sp. 75iv2 (Brown et al. 2011), Sphingomonas sp. strain SYK-6 (Masai et al. 2007) and Streptomyces viridosporus T7A (Ramachandra et al. 1988). Most likely, there are other bacteria with aromatic metabolizing capacities, which are yet to be identified. Recently, there has been an extensive search for more bacterial species from several natural or man-made environments, which exhibit certain lignin-degrading abilities thanks to the secretion of oxidoreductases, etherases and other enzymes (Picart et al. 2016; Taylor et al. 2012). Some of the reported organisms are, e.g. Klebsiella and Pseudomonas spp. from compost samples (Ravi et al. 2017), Cupriavidus basilensis B-8 (Shi et al. 2013) and Comamonas sp. B-9 from eroded bamboo slips (Chen et al. 2012), Bacillus pumilus and Bacillus atrophaeus from biodiversity-rich rainforest soil (Huang et al. 2013) and Trabulsiella sp. isolated from termite gut (Suman et al. 2016). It is essential not only to isolate these organisms, but also to characterize their inherent aromatic metabolism in order to assess their potential—as hosts or donor of pathways—for lignin valorization.

In the present study, our initial aim was to isolate and identify easily culturable bacterial species from sediments in the Baltic Sea, close to the wastewater stream of a sulfite pulp production plant in northern Sweden (Kramfors). In this environment, lignin-rich residuals were deposited and accumulated between 1907 and 1977 (Apler et al. 2014). Lignin-enriched wastewater effluents have a damaging effect to the aquatic ecosystems because of their toxic chemical compounds and also due to the recalcitrance of this polymer in natural environments (Berryman et al. 2004). Therefore bacterial species present in such polluted sediments are likely to possess aromatic metabolic capacities, and hence are interesting to investigate (Priyadarshinee et al. 2016). The second objective was to further examine the metabolism of aromatic compounds by the most interesting isolates. In particular, one of the isolates, identified as Pseudomonas sp. 9.1 was grown on six lignin model compounds (ferulate, p-coumarate, benzoate, syringate, vanillin and guaiacol) representing the main branches of the upper funneling catabolic pathways (Fig. 1) and specific growth rates, specific uptake rates and by-product formation from the aromatic compounds were quantified. The selected isolate, under the same initial aromatic compounds concentration, was found to behave quite differently from the previously studied P. putida KT2440 (Ravi et al. 2017) since it excreted several pathway intermediates. Further experiments were conducted with the excreted intermediates and the conversion rates of various upper funneling branches leading to β-ketoadipate pathway were assessed.

In the present study, a bacterial strain with extensive capacity for catabolism of aromatic compounds was isolated and physiologically characterized. The method of isolation, i.e. culture-dependent screening on either lignin or lignin-derived molecules from an interesting environment, is a widely used method to find lignin-utilizing prokaryotes (Tian et al. 2014) and proved successful also here.

Most of the bacterial isolates (five out of nine) from the enrichment cultures carried out in this study, belong to the γ-proteobacteria class, particularly to the genus Pseudomonas, the versatility of which to degrade and grow on lignin-related aromatic compounds and even technical lignins is widely acknowledged (Bandounas et al. 2011; Bugg and Rahmanpour 2015; Narbad and Gasson 1998). Three Gram-positive isolates from the Bacillaceae family were also detected by this growth-dependent method, namely Bacillus licheniformis, B. safensis and Lysinibacillus macroides. Many species of the genus Bacillus were previously found in deep-sea sediments in a similar screening carried out by Ohta et al. (2012), and most of them were also metabolically active toward lignin-related aromatic compounds. Finally, one Rhodococcus erythropolis strain, an actinomycete belonging to the high G+C Gram positive bacteria, was found as well. This is not surprising, since a number of rhodococci including R. erythropolis have been described among the most efficient bacterial lignin degraders (Ahmad et al. 2010) and are also able to metabolize the low-molecular weight compounds coming from lignin degradation (Taylor et al. 2012). Some of the bacterial isolates found in this work did not show ability to grow in liquid cultures with the model compounds utilized for their isolation (Additional file 1: Figs. S1 and S2). The initial growth on the screening plates might be due to the presence of intracellular storage compounds accumulated during the revitalization stage in rich medium.

Characterizing the aromatic metabolism, both in terms of catabolic pathways and the capacities of these pathways, is important to enable the design of efficient lignin conversion processes. The growth on low-molecular-weight lignin model compounds is relevant since the catabolism of such compounds is essential after, or along with, lignin mineralization (Bandounas et al. 2011). In the present study, six aromatic model compounds from the main different upper funneling pathways of lignin conversion were selected as carbon sources: vanillin, guaiacol and ferulate from the coniferyl branch; p-coumarate from the coumaryl branch; benzoate from the benzoyl branch and syringate from the sinapyl branch (Fig. 1). The coniferyl and coumaryl branches converge at protocatechuate (except for guaiacol which is converted to catechol), the benzoyl branch continues through catechol, whereas the sinapyl branch proceeds down to 3-O-methylgallic acid or even further to gallic acid. Protocatechuate and catechol enter the β-ketoadipate pathway ending with acetyl-CoA and succinyl-CoA, while 3-O-methylgallic acid/gallic acid undergoes ring cleavage and degrades to oxaloacetic acid and pyruvic acid. All these products either are the TCA (tricarboxylic acid) cycle intermediates or eventually enter the TCA cycle to promote cell growth. The isolate 9.1, tentatively identified as P. deceptionensis was able to take up four out of the six tested lignin model compounds as sole sources of carbon. The highest specific growth and uptake rates were measured for benzoate (Table 4), indicating that the benzoyl branch is the fastest of the upper funneling pathways in this isolate. The second fastest uptake occurred in the p-coumaryl branch with uptake rates of p-coumarate and 4-HBA around 2.9 mmol g _CDW ⁻¹  h⁻¹. The coniferyl branch operated at a somewhat lower flux rate, with uptake rates of ferulate, vanillate and vanillyl alcohol lower than 2 mmol g _CDW ⁻¹  h⁻¹. This might be due to the presence of phenylmethyl ether linkage in the coniferyl branch, which is comparatively difficult to breakdown (Okamura-Abe et al. 2016). No growth was obtained for syringate and guaiacol. The difficulty in metabolizing highly methoxylated substrates such as those present in the syringyl branch is most likely due to the complex enzymatic systems required for its demethylation. The O-demethylase systems for dimethoxylated compounds are well described in the case of Sphingobium sp. SYK-6, Acetobacterium woodii, Acetobacterium dehalogenans and Moorella thermoacetica (Berman and Frazer 1992; Kaufmann et al. 1998; Naidu and Ragsdale 2001). They consist of two methyl transferases which convey the methyl groups from the aromatic substrate (e.g. veratrol, syringate) to an accepting cofactor like tetrahydrofolate (H₄F), often with the involvement of a corrinoid protein. These systems usually show relatively high substrate specificity, and also require continuous supply and enzymatic recycling of the cofactor; moreover, when a corrinoid protein is involved, spontaneous oxidation of its cobalt group can take place, and ATP-consuming enzymatic regeneration of this oxidized catalyst will be necessary (Siebert et al. 2005). Additionally, the demethylation reactions can result in the formation of formaldehyde or formic acid (Masai et al. 2007; Overhage et al. 1999), toxic intermediates that require further processing (Hibi et al. 2005), which can contribute to the slower metabolism of these compounds (Additional file 1: Figs. S3 and S4).

There was a short lag phase of approximately 15 h for ferulate, p-coumarate and benzoate (Fig. 2), necessary for adaption to the new carbon source and expression of required enzymes after prior growth on glucose (Kurosawa et al. 2015). A much longer lag phase, around 100 h, for growth on vanillin (Fig. 2) and no growth on syringate for 200 h, was observed. The antimicrobial effect of both compounds is well documented for many organisms. The concentration of vanillin and syringate required to inhibit the growth the Escherichia coli LY01 by 50% is 0.9 and 1.6 g/L respectively (Zaldivar et al. 1999). The mechanism of inhibition can be either bacteriostatic or bactericidal. The increased uptake of nucleic acid stain upon exposure of E. coli to vanillin suggests that the integrity and stability of the cell membrane was affected, leading to cell lysis (Fitzgerald et al. 2004). In another study, the addition of syringic acid at the concentration of IC₈₀ (80% inhibitory concentration), proved to destroy the membrane integrity by increasing membrane leakage by three fold (Zaldivar and Ingram 1999). However, in our case, no major cell lysis (i.e. no decrease in OD) was observed when exposed to vanillin or syringate, which suggests that the inhibition was bacteriostatic. A quite long lag phase before growth on vanillin, might explain the time required by the organism to repair the cell damage and overcome the inhibition (Rolfe et al. 2012).

Interestingly, Pseudomonas sp. isolate 9.1 while growing on some model compounds excreted certain pathway intermediates. This contrasts to previous observations with P. putida strains (Ravi et al. 2017). It appears that isolate 9.1 is not regulated to maintain the same flux throughout the pathway to the same extent as P. putida. With the exception of cis,cis-muconate, all the excreted intermediates were later consumed, thereby enabling the complete utilization of the carbon source, although in a di- or tri-auxic growth pattern. Cis,cis-muconate and catechol were excreted during growth on benzoate, but while catechol was subsequently taken up, cis,cis-muconate remained unconsumed (Fig. 2c). Possibly Pseudomonas sp. isolate 9.1 lacks or has a defective version of the gene encoding the transporter involved in the uptake of cis,cis-muconate, which in Acinetobacter and other Pseudomonas strains has been identified as mucK (Williams and Shaw 1997), a member of the family 15 of MFS transporters (Pao et al. 1998). It is not clear how the accumulated cis,cis-muconate is excreted outside of the cell without a functional transporter, but there may be other transporters able to move this compound across the cell membrane with lower specificity. Cis,cis-muconate can be used as a precursor to adipic acid, one of the monomers in nylon-6,6 (Vardon et al. 2015) and has therefore attracted quite some interest. Bioconversion of benzoate to cis,cis-muconate was explored in P. putida KT2440-JD1 (van Duuren et al. 2012). Also in this P. putida strain, the maximum uptake rate of benzoate was higher than the production rate of cis,cis-muconate, which is the reason for the excretion of benzoate derivatives in the cells. Excessive uptake of benzoate is also a reason for the excretion of catechol, which reduces the expression of the ben operon leading to the decreased conversion of benzoate to cis,cis-muconate (van Duuren et al. 2012).

Accumulation of catechol in the culture media might be toxic to the organism since catechol is unstable at pH 7 and produces reactive oxygen species. The oxidized species spontaneously form colored polymers (Subramanian and Worden 1993; Sudarsan et al. 2016; Yang et al. 2014), which give experimental problems to study catechol as a sole source of carbon in growth experiments, also in the present work.

Somewhat unexpectedly, vanillyl alcohol was excreted during growth of Pseudomonas sp. isolate 9.1 on vanillin (Fig. 2 vanillin). As vanillin is a potential inhibitor to many microorganisms, the conversion of vanillin to less toxic intermediates is quite common (Gallage and Møller 2015). In bacteria, especially pseudomonads, vanillin is normally converted to vanillate (Graf and Altenbuchner 2014; Ravi et al. 2017). However, under growing conditions, P. fluorescens B56 was able to convert vanillin to vanillate with a small amount of vanillyl alcohol being excreted (Asm et al. 1988). In addition, a genetically modified Pseudomonas strain (vanillin dehydrogenase deletion mutant) was able to produce vanillyl alcohol via ferulic acid and vanillin, when eugenol was given as a sole carbon source (Takasago Perfumery Co Ltd 1993). Although the intention of genetic modification was to produce vanillin from eugenol, the strain further converted some amount of vanillin to vanillyl alcohol, which proves the existence of such activity under certain conditions.

The conversion of vanillin to vanillyl alcohol is more common as a part of the detoxification process in eukaryotes such as Phanerochaete chrysosporium or Saccharomyces cerevisiae, (Nguyen et al. 2015; Stentelaire et al. 1997). The uptake rate of vanillin by isolate 9.1 was 3.19 mmol g _CDW ⁻¹  h⁻¹, whereas the uptake of vanillate was only 1.95 mmol g _CDW ⁻¹  h⁻¹. The excess uptake of vanillin was apparently channeled to vanillyl alcohol, which was subsequently giving rise to a diauxic growth pattern.

Another observation was that Pseudomonas sp. isolate 9.1 had a relatively stable stationary phase after the carbon source was depleted (Figs. 2, 3). This is in contrast to our previous work with P. putida KT2440, in which case there was a rapid decrease in the biomass concentration directly after the depletion of the carbon source without a stable stationary phase (Ravi et al. 2017).

In conclusion, the isolate 9.1, tentatively identified as Pseudomonas deceptionensis based on gyrB alignment, appears to possess most of the major upper funneling pathways necessary for the conversion of lignin-related aromatic compounds. Growth on model compounds does not mean that the organism will necessarily convert lignin or depolymerized lignin. However, it is a prerequisite for such conversion to occur.

Further work will show if this isolate or other bacterial strains are able to metabolize or partially convert different types of depolymerized lignin, which is necessary for the establishment of efficient processes in the future biorefineries, paving the way for a complete exploitation of the forestry and agricultural resources.