Lignin is a major component of lignocellulosic biomass and as such, it is processed in enormous amounts in the pulp and paper industry worldwide. In such industry it mainly serves the purpose of a fuel to provide process steam and electricity, and to a minor extent to provide low grade heat for external purposes. Also from other biorefinery concepts, including 2nd generation ethanol, increasing amounts of lignin will be generated. Other uses for lignin - apart from fuel production - are of increasing interest not least in these new biorefinery concepts. These new uses can broadly be divided into application of the polymer as such, native or modified, or the use of lignin as a feedstock for the production of chemicals. The present review focuses on the latter and in particular the advances in the biological routes for chemicals production from lignin. Such a biological route will likely involve an initial depolymerization, which is followed by biological conversion of the obtained smaller lignin fragments. The conversion can be either a short catalytic conversion into desired chemicals, or a longer metabolic conversion. In this review, we give a brief summary of sources of lignin, methods of depolymerization, biological pathways for conversion of the lignin monomers and the analytical tools necessary for characterizing and evaluating key lignin attributes.

Starting from mature vegetable compost, four bacterial strains were selected using a lignin-rich medium. 16S ribosomal RNA identification of the isolates showed high score similarity with Pseudomonas spp. for three out of four isolates. Further characterization of growth on mixtures of six selected lignin model compounds (vanillin, vanillate, 4-hydroxybenzoate, p-coumarate, benzoate, and ferulate) was carried out with three of the Pseudomonas isolates and in addition with the strain Pseudomonas putida KT2440 from a culture collection. The specific growth rates on benzoate, p-coumarate, and 4-hydroxybenzoate were considerably higher (0.26–0.27 h−1) than those on ferulate and vanillate (0.21 and 0.22 h−1), as were the uptake rates. There was no direct growth of P. putida KT2440 on vanillin, but instead, vanillin was rapidly converted into vanillate at a rate of 4.87 mmol (gCDW h)−1 after which the accumulated vanillate was taken up. The growth curve reflected a diauxic growth when mixtures of the model compounds were used as carbon source. Vanillin, 4-hydroxybenzoate, and benzoate were preferentially consumed first, whereas ferulate was always the last substrate to be taken in. These results contribute to a better understanding of the aromatic metabolism of P. putida in terms of growth and uptake rates, which will be helpful for the utilization of these bacteria as cell factories for upgrading lignin-derived mixtures of aromatic molecules.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-017-8211-y) contains supplementary material, which is available to authorized users.

ABSTRACTThe obligate predatorBdellovibrio bacteriovorusHD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 ofB. bacteriovorusHD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZBd). The primary structure of PhaZBdsuggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZBdhas been extracellularly produced using different hypersecretor Tol-pal mutants ofEscherichia coliandPseudomonas putidaas recombinant hosts. The recombinant PhaZBdhas been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZBdis an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.

The phaZ ( Sex ) gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T (m)) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine-histidine-carboxylic acid in the active site. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.

Bacterial strains were isolated from the sediments of the Baltic Sea using ferulic acid, guaiacol or a lignin-rich softwood waste stream as substrate. In total nine isolates were obtained, five on ferulic acid, two on guaiacol and two on a lignin-rich softwood stream as a carbon source. Three of the isolates were found to be Pseudomonas sp. based on 16S rRNA sequencing. Among them, isolate 9.1, which showed the fastest growth in defined M9 medium, was tentatively identified as a Pseudomonas deceptionensis strain based on the gyrB sequencing. The growth of isolate 9.1 was further examined on six selected lignin model compounds (ferulate, p-coumarate, benzoate, syringate, vanillin and guaiacol) from different upper funneling aromatic pathways and was found able to grow on four out of these six compounds. No growth was detected on syringate and guaiacol. The highest specific growth and uptake rates were observed for benzoate (0.3 h−1 and 4.2 mmol gCDW−1 h−1) whereas the lowest were for the compounds from the coniferyl branch. Interestingly, several pathway intermediates were excreted during batch growth. Vanillyl alcohol was found to be excreted during growth on vanillin. Several other intermediates like cis,cis-muconate, catechol, vanillate and 4-hydroxybenzoate from the known bacterial catabolic pathways were excreted during growth on the model compounds.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0563-x) contains supplementary material, which is available to authorized users.

BackgroundLignin is a potential feedstock for microbial conversion into various chemicals. However, the microbial degradation rate of native or technical lignin is low, and chemical depolymerization is needed to obtain reasonable conversion rates. In the current study, nine bacterial strains belonging to the Pseudomonas and Rhodococcus genera were evaluated for their ability to grow on alkaline-treated softwood lignin as a sole carbon source.ResultsPseudomonas fluorescens DSM 50090 and Rhodococcus opacus DSM1069 showed the best growth of the tested species on plates with lignin. Further evaluation of P. fluorescens and R. opacus was made in liquid cultivations with depolymerized softwood Kraft lignin (DL) at a concentration of 1 g/L. Size-exclusion chromatography (SEC) showed that R. opacus consumed most of the available lower-molecular weight compounds (approximately 0.1–0.4 kDa) in the DL, but the weight distribution of larger fractions was almost unaffected. Importantly, the consumed compounds included guaiacol—one of the main monomers in the DL. SEC analysis of P. fluorescens culture broth, in contrast, did not show a large conversion of low-molecular weight compounds, and guaiacol remained unconsumed. However, a significant shift in molecular weight distribution towards lower average weights was seen after cultivation with P. fluorescens.ConclusionsRhodococcus opacus and P. fluorescens were identified as two potential microbial candidates for the conversion/consumption of base-catalyzed depolymerized lignin, acting on low- and high-molecular weight lignin fragments, respectively. These findings will be of relevance for designing bioconversion of softwood Kraft lignin.Electronic supplementary materialThe online version of this article (10.1186/s13068-019-1397-8) contains supplementary material, which is available to authorized users.

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The ascomycin-producer strain Streptomyces ascomycinicus has been proven to be an extracellular poly(R)-3-hydroxybutyrate (PHB) degrader. The fkbU gene, encoding a PHB depolymerase (PhaZSa), has been cloned in E. coli and Rhodococcus sp. T104 strains for gene expression. Gram-positive host Rhodococcus sp. T104 was able to produce and secrete to the extracellular medium an active protein form. PhaZSa was purified by two hydrophobic interaction chromatographic steps, and afterwards was biochemically as well as structurally characterized. The enzyme was found to be a monomer with a molecular mass of 48.4 kDa, and displayed highest activity at 45°C and pH 6, thus being the first PHB depolymerase from a gram-positive bacterium presenting an acidic pH optimum. The PHB depolymerase activity of PhaZSa was increased in the presence of divalent cations due to non-essential activation, and also in the presence of methyl-β-cyclodextrin and PEG 3350. Protein structure was analyzed, revealing a globular shape with an alpha-beta hydrolase fold. The amino acids comprising the catalytic triad, Ser131-Asp209-His269, were identified by multiple sequence alignment, chemical modification of amino acids and site-directed mutagenesis. These structural results supported the proposal of a three-dimensional model for this depolymerase. PhaZSa was able to degrade PHB, but also demonstrated its ability to degrade films made of PHB, PHBV copolymers and a blend of PHB and starch (7∶3 proportion wt/wt). The features shown by PhaZSa make it an interesting candidate for industrial applications involving PHB degradation.