Biodegradation of kraft lignin by a bacterial strain Comamonas sp. B-9 isolated from eroded bamboo slips

Aims: The aim was to obtain evidences for lignin degradation by unicellular bacterium Comamonas sp. B‐9. Methods and Results: Comamonas sp. B‐9 was inoculated into kraft lignin‐mineral salt medium (KL‐MSM) at pH 7·0 and 30°C for 7 days of incubation. The bacterial growth, chemical oxygen demand (COD) reduction, secretion of ligninolytic enzymes and productions of low‐molecular‐weight compounds revealed that Comamonas sp. B‐9 was able to degrade kraft lignin (KL). COD in KL‐MSM reduced by 32% after 7 days of incubation. The maximum activities of manganese peroxidase (MnP) of 2903·2 U l^?1 and laccase (Lac) of 1250 U l^?1 were observed at 4th and 6th day, respectively. The low‐molecular‐weight compounds such as ethanediol, 3, 5‐dimethyl‐benzaldehyde and phenethyl alcohol were formed in the degradation of KL by Comamonas sp. B‐9 based on GC‐MS analysis. Conclusions: This study confirmed that Comamonas sp. B‐9 could utilize KL as a sole carbon source and degrade KL to low‐molecular‐weight compounds. Significance and Impact of the Study: Comamonas sp. B‐9 may be useful in the utilization and bioconversion of lignin and lignin‐derived aromatic compounds in biotechnological applications. Meanwhile, using Comamonas sp. B‐9 in treatment of wastewater in pulp and paper industry is a meaningful work.     

Depolymerization and decolorization of kraft lignin by bacterium Comamonas sp. B-9

There is no commercial or industrial-scale process for the remediation of black liquor using microorganisms to date. One of the most important causes is that most microorganisms are not able to use lignin as their principal metabolic carbon or energy source. The bacterial strain Comamonas sp. B-9 has shown remarkable ability to degrade kraft lignin and decolorize black liquor using lignin as its principal metabolic carbon and energy source. This report looks at the depolymerization and decolorization of kraft lignin by Comamonas sp. B-9. The degradation, decolorization, and total carbon removal reached 45, 54, and 47.3 %, respectively, after 7 days treatment. Comamonas sp. B-9 was capable of depolymerizing kraft lignin effectively as analyzed by gel permeation chromatography and decolorization via degrading benzene ring structures as shown using Fourier transform infrared spectroscopy analysis.     

Fast degradation of kraft lignin by bacteria

Summary Lignin degrading bacteria were isolated directly by an enrichment culture technique using an industrial kraft lignin (Indulin AT) as the sole carbon source. The lignin degrading ability of these isolates was assayed in pure cultures. One strain (Aeromonas sp.) had degraded 98% of the lignin (1 g/l) after 5 days of incubation. Different genera have been identified including Corynebacterium, Agrobacterium, Pseudomonas, Aeromonas, but also Klebsiella and Enterobacter. These strains were also able to assimilate different phenolic compounds considered as lignin related simple monomers.     

新生儿血培养检出潘多拉菌一例

潘多拉菌是一种革兰阴性非发酵菌,隶属伯克霍尔德菌属。国内潘多拉菌感染鲜有临床报道。本实验室在1例新生儿血培养中检出一株革兰阴性杆菌,经生化、药敏试验及16S rRNA基因测序等方法鉴定,确定是潘多拉菌属。     

Polysaccharide synthesis by Phanerochaete chrysosporium during degradation of kraft lignin

Summary Phanerochaete chrysosporium (Sporotrichum pulverulentum) produced an extracellular glucan type polysaccharide when grown in a chemostat under nitrogen limitation. When cells were transferred to a standing mode of cultivation in the presence of excess glucose (6 gl^–1), the amount of non-glucose total carbohydrates in the culture increased from 0.58 gl^–1 to 1.76 gl^–1 during 15 day experiments. The change in total carbohydrates was due to an increase in extracellular and cell-bound glucan type polysaccharide. This increase occured simultaneously with formation of mycelial mats and appearance of ligninolytic activity. When the cultures were agitated under atmospheric oxygen rather than 100% O₂, their non-glucose total carbohydrate content increased to 2.15 gl^–1 in 4 days. The excess polysaccharide formation had an inhibitory effect on lignin degradation as more lignin was degraded by cells with lower polysaccharide content. The lignin that was associated with cells after the degradation had stopped could be further degraded by new active cells.     

Regioselective carboxylation of 1,3-dihydroxybenzene by 2,6-dihydroxybenzoate decarboxylase of Pandoraea sp. 12B-2

We found a bacterium, Pandoraea sp. 12B-2, of which whole cells catalyzed not only the decarboxylation of 2,6-dihydroxybenzoate but also the regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate. The whole cells of Pandoraea sp. 12B-2 also catalyzed the regioselective carboxylation of phenol and 1,2-dihydroxybenzene to 4-hydroxybenzoate and 2,3-dihydroxybenzoate, respectively. The molar conversion ratio of the carboxylation reaction depended on the concentration of KHCO₃ in the reaction mixture. Only 5 or 48 % of 1,3-dihydroxybenzene added was converted into 2,6-dihydroxybenzoate in the presence of 0.1 M or 3 M KHCO₃, respectively. The addition of acetone to the reaction mixture increased the initial rate of the carboxylation reaction, but the final molar conversion yield reached almost the same value. When the efficient production of 2,6-dihydroxybenzoate was optimized using the whole cells of Pandoraea sp. 12B-2, the productivity of 2,6-dihydroxybenzoate topped out at 1.43 M, which was the highest value so far reported. No formation of any other products was observed after the carboxylation reaction.     

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (mol xylose min^–1 ml^–1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.     

Isolation and some properties of newly isolated oxalate-degrading Pandoraea sp. OXJ-11 from soil

AIMS: To isolate and characterize an oxalate-degrading Pandoraea sp. OXJ-11. METHODS AND RESULTS: A new bacterium Pandoraea sp. OXJ-11 was isolated from soil samples, which can grow in the medium with oxalate as the sole carbon and energy source. The isolate OXJ-11 is Gram-negative straight rod. It occurs singly and is motile by means of a double polar flagellum. Catalase is positive and nitrate is not reduced. It grows aerobically and the optimum growth temperature and the optimum pH are at 30 degrees C and pH 6.0, respectively. The polyphasic taxonomic data along with 16S rRNA sequence comparison demonstrate that the isolate OXJ-11 should belong to the genus Pandoraea and represent a new member in this family. CONCLUSIONS: Oxalate could be degraded and the oxalate-degrading enzyme activity was detected when the isolate OXJ-11 grew in the medium with oxalate as carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxalate-degrading Pandoraea sp. OXJ-11 would be beneficial to the potential application in the control of sclerotinia stem rot in economically important plants caused by fungus Sclerotinia sclerotiorum, and in making plants resistant to the white mold disease by oxalate-degrading enzyme transgene.     

Characterization and genomic analysis of kraft lignin biodegradation by the beta-proteobacterium Cupriavidus basilensis B-8

Background

Lignin materials are abundant and among the most important potential sources for biofuel production. Development of an efficient lignin degradation process has considerable potential for the production of a variety of chemicals, including bioethanol. However, lignin degradation using current methods is inefficient. Given their immense environmental adaptability and biochemical versatility, bacterial could be used as a valuable tool for the rapid degradation of lignin. Kraft lignin (KL) is a polymer by-product of the pulp and paper industry resulting from alkaline sulfide treatment of lignocellulose, and it has been widely used for lignin-related studies.

Results

Beta-proteobacterium Cupriavidus basilensis B-8 isolated from erosive bamboo slips displayed substantial KL degradation capability. With initial concentrations of 0.5–6 g L^-1, at least 31.3% KL could be degraded in 7 days. The maximum degradation rate was 44.4% at the initial concentration of 2 g L^-1. The optimum pH and temperature for KL degradation were 7.0 and 30°C, respectively. Manganese peroxidase (MnP) and laccase (Lac) demonstrated their greatest level of activity, 1685.3 U L^-1 and 815.6 U L^-1, at the third and fourth days, respectively. Many small molecule intermediates were formed during the process of KL degradation, as determined using GC-MS analysis. In order to perform metabolic reconstruction of lignin degradation in this bacterium, a draft genome sequence for C. basilensis B-8 was generated. Genomic analysis focused on the catabolic potential of this bacterium against several lignin-derived compounds. These analyses together with sequence comparisons predicted the existence of three major metabolic pathways: β-ketoadipate, phenol degradation, and gentisate pathways.

Conclusion

These results confirmed the capability of C. basilensis B-8 to promote KL degradation. Whole genomic sequencing and systematic analysis of the C. basilensis B-8 genome identified degradation steps and intermediates from this bacterial-mediated KL degradation method. Our findings provide a theoretical basis for research into the mechanisms of lignin degradation as well as a practical basis for biofuel production using lignin materials.     

Fungal degradation of kraft lignin and lignin sulfonates prepared form synthetic 14C-lignins

Kraft lignins (KL), bleached kraft lignins (BKL), and lignin sulfonates (LS) were prepared from synthetic ¹⁴C-lignins labeled in the aromatic nuclei or in the propyl side chains. These and control lignins (CL) were incubated with the lignin-decomposing white-rot fungus, Phanerochaete chrysosporium Burds., in a defined culture medium containing cellulose as growth substrate. Decomposition was monitored by measuring the ¹⁴CO₂ evolved. Average percentages of the [ring-¹⁴C]- and [side chain-¹⁴C]-lignins, respectively, recovered as ¹⁴CO₂ at the cessation of ¹⁴CO₂ evolution were: KL, 41 and 31; BKL, 42 and 26; LS, 28 and 21; and CL, 26 and 24. Gel permeation chromatography of radiolabeled materials extracted from spent cultures showed that substantial degradation to nonvolatile products had occurred. The polymeric components in the extracts were further degraded in fresh cultures. These results indicate that industrial lignins are significantly bioalterable, and that under favorable conditions industrial lignins are substantially biodegradable.     

Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor

In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.     

Pedobacter seoulensis sp. nov., isolated from soil of a bamboo field

A Gram-stain negative, strictly aerobic, motile by gliding, rod-shaped and yellow pigmented strain THG-G12^T was isolated from soil of a bamboo field in Seoul, Republic of Korea. Strain THG-G12^T was observed to grow well at 20–28 °C and pH 7.0–7.5 in the absence of NaCl on nutrient agar. Based on 16S rRNA gene sequence comparisons, strain THG-G12^T was found to be most closely related to Pedobacter ginsengisoli Gsoil 104^T (97.5 % sequence similarity), Pedobacter steynii WB2.3-45^T (97.4 %), Pedobacter metabolipauper WB2.3-71^T (97.2 %), Pedobacter nyackensis NWG-II14^T (97.2 %), Pedobacter caeni LMG 22862^T (97.1 %) and Pedobacter duraquae WB2.1-25^T (97.0 %), but DNA relatedness between strain THG-G12^T and its phylogenetically closest neighbours was below 9.5 %. The G+C content of the genomic DNA was determined to be 39.9 mol%. The only isoprenoid quinone detected in strain THG-G12^T was menaquinone-7 (MK-7). The major component in the polyamine pattern was sym-homospermidine. The major polar lipids were found to be phosphatidylethanolamine, unidentified phosphoglycolipids, unidentified aminophosphoglycolipids, unidentified aminolipids and unidentified lipids. Strain THG-G12^T showed the presence of two ceramide phosphorylethanolamines (CerPE-2′ and CerPE-2″), dihydrosphingosines and an unidentified ceramide as the major ceramide. The major fatty acids were identified as summed feature 3 (as defined by the MIDI system; C_16:1 ω7c and/or C_16:1 ω6c) and iso-C_15:0. These data support the affiliation of strain THG-G12^T to the genus Pedobacter. The results of physiological and biochemical tests enabled strain THG-G12^T to be differentiated genotypically and phenotypically from the recognized species of the genus Pedobacter. Therefore, the novel isolate represents a novel species, for which the name Pedobacter seoulensis sp. nov. is proposed, with THG-G12^T as the type strain (=KACC 17529^T =JCM 19363^T).     

Identification of low molecular weight aromatic compounds by gas chromatography–mass spectrometry (GC–MS) from kraft lignin degradation by three Bacillus sp.

Three bacterial strains identified as Paenibacillus sp., Aneurinibacillus aneurinilyticus and Bacillus sp. have been shown to decolourise kraft lignin in 6 days of incubation. The release of low molecular weight aromatic compounds by these bacterial strains during degradation of kraft lignin was analysed by GC–MS analysis. The total ion chromatograph (TIC) of ethyl acetate extract from kraft lignin sample inoculated by Paenibacillus sp. was similar to control except some minor changes in the chromatographic profile indicating incapability of this bacterium to modify kraft lignin. On the other hand the TIC of ethyl acetate extract from kraft lignin inoculated by A. aneurinilyticus and Bacillus sp. caused formation of several aromatic lignin-related compound that were not present in the extract of control. The compounds identified in extract of the sample degraded by A. aneurinilyticus were guaiacol, acetoguiacone, gallic acid and ferulic acid while t-cinnamic acid, 3,4,5 trimethoxy benzaldehyde, and ferulic acid by Bacillus sp. indicating oxidization of coniferylic (G units) and sinapylic (S units) alcohol of lignin polymer. Differences between the identified compounds from different bacterial treatment were strain-specific. Among the identified aromatic compounds, ferulic acid and 3,4,5-trimethoxy benzaldehyde could be useful to the industry of preservatives, aromas and perfumes.     

Biotransformation of kraft lignin fractions by Serratia marcescens

The ability of a Serratia marcescens strain to transform ethyl acetate soluble (EASF) and insoluble (EAIF) fractions of kraft pine lignin, was studied, In media containing EASF or EAIF and 0.1% glucoes, the bacterium degraded 15 and 9% respectively, as shown by ultraviolet spectrophotometry. The greates percentages degradation, 48 and 43% were obtained when S. marcescens grew on neutral (N-EASF) and acid (A-EASFF) compnents of the EASF of kraft lignin     

Degradation of lignin and lignin derivatives by Acinetobacter sp.

An Acinetobacter sp. utilized 2-methoxy-4-formylphenoxyacetic acid, dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin as sole carbon source. It also degraded ¹⁴C-labelled DHP lignin and teakwood lignin. Vanillic acid, protocatechuic acid and catechol were separated from 2-methoxy-4-formylphenoxyacetic acid grown cultures. Both protocatechuic acid and catechol were formed from dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin. On the dimeric lignin model substances this Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase.     

Enhanced degradation of gamma-irradiated lignocelluloses by a new xylanolytic Flavobacterium sp. isolated from soil

An extracellular chitosanase produced by Rhodotorula gracilis CFR-1 that catalyses a limited degradation of chitosan with no detectable generation of glucosamine or reducing groups was identified. Ultracentrifugation, polyacrylamide gel electrophoresis and gel permeation studies suggest that chitosan of average molecular mass 36000 Da was reduced by the enzymic catalysis to nearly one-fourth this size without further hydrolysis of the products. The enzyme, produced constitutively by this yeast, was partially purified and some of its properties were studied.     

Anaerobic degradation of 3-hydroxypropanoate by a newly isolated Clostridium sp.

A strain (ToHP1) of anaerobic bacteria which degrades 3-hydroxypropanoate was isolated in pure culture from the sludge of an anaerobic digestor at a Tokyo sewage treatment facility. The strain grew by degrading 3-hydroxypropanoate, lactate, and pyruvate to propionate and acetate. It also grew auxotrophically by degrading 2-hydroxybutanoate to butyrate and propionate in the presence of acetate. Cells were lemon-shaped rods; 1.4 to 3.6 μm in length and 0.5 to 1.1 μm in width. Spores with a diameter of 0.8 to 1.1 μm were formed subterminally. The strain occurred singly or in pairs. It stained gram-positive. The strain grew optimally at 30 to 35°C and ca. pH 7.2. It required yeast extract for growth. The strain did not utilize nitrate, sulfate, sulfite, thiosulfate, or elemental sulfur as an electron acceptor. The guanine plus cytosine content of the DNA was 43 mol%. Strain ToHP1 was identified as a member of the genus Clostridium.     

Rapid degradation of the triazinone herbicide metamitron by a Rhodococcus sp. isolated from treated soil

N.R. PAREKH, A. WALKER, S.J. ROBERTS AND S.J. WELCH. 1994. Seven bacterial isolates which degraded the herbicide metamitron (3-methyl-4-amino-6-phenyl-1,2,4-triazin-5-one) were obtained from field-enhanced soil by liquid enrichment culture. All isolates appeared to be identical and a representative, 0246b, was identified as a Rhodococcus sp. by cell wall and fatty acid analyses. This isolate degraded metamitron as the sole source of carbon within 24 h at 25C and this is the first report of a bacterium capable of growing with metamitron as the sole source of carbon. Metamitron was degraded less rapidly when it was the sole source of both carbon and nitrogen. The rate and extent of degradation was affected by the presence and type of additional sources of carbon and nitrogen in the culture medium. In studies with [¹⁴C]-phenyl-labelled metamitron Rhodococcus sp. 0246b partly mineralized the phenyl ring.