Delignification and enhanced gas release from soil containing lignocellulose by treatment with bacterial lignin degraders.

AIMS: The aim of the study was to isolate bacterial lignin-degrading bacteria from municipal solid waste (MSW) soil, and to investigate whether they could be used to delignify lignocellulose-containing soil, and enhance methane release. METHODS AND RESULTS: A set of 20 bacterial lignin degraders, including 11 new isolates from MSW soil, were tested for delignification and phenol release in soil containing 1% pine lignocellulose. A group of seven strains were then tested for enhancement of gas release from soil containing 1% lignocellulose in small-scale column tests. Using an aerobic pretreatment, aerobic strains such as Pseudomonas putida showed enhanced gas release from the treated sample, but four bacterial isolates showed 5-10-fold enhancement in gas release in an in situ experiment under microanaerobic conditions: Agrobacterium sp., Lysinibacillus sphaericus, Comamonas testosteroni and Enterobacter sp. CONCLUSIONS: The results show that facultative anaerobic bacterial lignin degraders found in landfill soil can be used for in situ delignification and enhanced gas release in soil containing lignocellulose. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the feasibility of using an in situ bacterial treatment to enhance gas release and resource recovery from landfill soil containing lignocellulosic waste.

Isolation of bacterial strains able to metabolize lignin from screening of environmental samples.

AIMS: To develop a method to detect bacteria from environmental samples that are able to metabolize lignin. METHODS AND RESULTS: A previously developed UV-vis assay method for lignin degradation activity has been developed for use as a spray assay on agar plates. Nine mesophilic strains were isolated using this method from woodland soil incubated in enrichment cultures containing wheat straw lignocellulose: four Microbacterium isolates, two Micrococcus isolates, Rhodococcus erythropolis (all Actinobacteria) and two Ochrobactrum isolates (Alphaproteobacteria). Three thermotolerant isolates were isolated from the same screening method applied at 45°C to samples of composted wheat straw from solid-state fermentation: Thermobifida fusca and two isolates related to uncharacterized species of Rhizobiales and Sphingobacterium (Bacteroidetes), the latter strain showing tenfold higher lignin degradation activity than other isolates. The isolated strains were able to depolymerize samples of size-fractionated high molecular weight and low molecular weight Kraft lignin, and produced low molecular weight metabolites oxalic acid and protocatechuic acid from incubations containing wheat straw lignocellulose. CONCLUSIONS: A new method for the isolation of bacteria able to metabolize lignin has been developed, which has been used to identify 12 bacterial isolates from environmental sources. The majority of isolates cluster into the Actinobacteria and the Alphaproteobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Lignin-degrading bacterial strains could be used to convert lignin-containing feedstocks into renewable chemicals and to identify new bacterial lignin-degrading enzymes.

The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism.

2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 A resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the alpha/beta hydrolase fold family, comprising eight beta-strands interconnected by loops and helices. The 2.8 A resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.