Screening for lignin degrading bacteria by means of 14C-labelled lignins

Several Nocardia and Pseudomonas spp., as well as some unidentified bacteria, isolated from lake water containing high loads of waste lignin, were tested for their capacity to release ¹⁴CO₂ from specifically ¹⁴C-labelled dehydropolymer of coniferyl alcohol (DHP) or corn stalk lignins. The bacteria were selected according to their ability to degrade phenolic compounds. However, only some of them could release significant amounts of ¹⁴CO₂ from the labelled lignin. The tested Nocardia spp. were more active than the Pseudomonas spp. and the unidentified bacteria. The most active strains belonged to N. autotrophica. These strains released CO₂ significantly from the methoxyl group and transformed the other carbons from the phenylpropane skeleton of lignin also into CO₂. Other less demethylating strains also released little CO₂ from the other carbons of the lignin molecule. From corn stalk materials which were specifically labelled in the lignin part only small amounts of labelled CO₂ were released.Non-Common-Abbreviation Used DHP dehydropolymers of coniferyl alcohol     

Colonization and degradation of rubber pieces by Nocardia sp.

The growth of a Nocardia sp. occurs essentially on the insoluble rubber substrate and the cells are tightly bound to the rubber in the initial stage of the growth in spite of vigorous stirring of the cultures. The colonization of rubber pieces was followed by staining with Schiff reagent, and it was revealed that not only the thickness of rubber pieces, but also their length and width greatly influenced microbial colonization and degradation of natural rubber products. Among rubber pieces of various shapes, long strips were most rapidly covered by many microbial colonies and experienced the highest rate of rubber degradation. The rate of degradation (expressed by % weight loss) of the long strips of rubber was a linear function of surface area per unit weight of rubber. Thin and wide films of rubber were also rapidly colonized and degraded, while the colonization and degradation of short and narrow pieces were substantially slower and less extensive.     

Decomposition of 14C-labelled lignin,holocellulose and lignocellulose by mycorrhizal fungi

Five different species of known ecto-mycorrhizal fungi: Cenococcum geophilum, Amanita muscaria, Tricholoma aurantium, Rhizopogon luteolus and Rhizopogon roseolus were studied for their ability to metabolize the major components of plant cell walls. All strains were able to decompose ¹⁴C-labelled plant lignin, ¹⁴C-lignocellulose and ¹⁴C-DHP-lignin at a rate which was lower than the one observed for the known white rot fungi Heterobasidion annosum and Sporotrichum pulverulentum. Also ¹⁴C-(U)-holocellulose was relatively less degradable for the mycorrhizal fungi than for the white rotters. On the other hand, aromatic monomers like ¹⁴C-vanillic acid were decomposed to a much higher extent by two species of mycorrhizal fungi compared to the activity observed for Heterobasidion annosum. The results of the experiments reveal that these stains of mycorrhizal fungi are well able to utilize the major components of plant material and thus can contribute to litter decomposition in the forest floor.     

Degradation of 14C-labelled lignin and dehydropolymer of coniferyl alcohol by ericoid and ectomycorrhizal fungi

The ability of ericoid and ectomycorrhizal fungi to utilize ¹⁴C-labelled lignin and O¹⁴CH₃-labelled dehydropolymer of coniferyl alcohol as sole C sources has been assessed in pure culture studies. The results indicate that ericoid mycorrhizal fungi are more effective in degrading lignin than ectomycorrhizal fungi. Amongst the ectomycorrhizal fungi the facultative mycorrhizal fungus Paxillus involutus degraded lignin more readily than those which are normally considered to be obligately mycorrhizal fungi such as Suillus bovinus and Rhizopogon roseolus. The importance of these lignin degrading capabilities is discussed in relation to the predominance of specific mycorrhiza forms along a gradient of increasing organic matter and hence lignin content of soil.     

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.     

Purification,characterization and regulation of a monomeric l-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239

In Nocardia sp. 239 d-phenylalanine is converted into l-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent l-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on l- or d-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and l-phenylalanine. Highest rates of l-phenylalanine dehydrogenase production were observed with increasing ratios of l-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from l- and d-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42 000 and consisted of a single subunit; it showed activity with l-phenylalanine, phenylpyruvate, 4-hydroxyphenyl-pyruvate, indole-3-pyruvate and -ketoisocaproate, but not with imidazolepyruvate, d-phenylalanine and other l-amino acids tested. Maximum activities with phenylpyruvate (310 mol min^-1 mg^-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPT hexulose-6-phosphate isomerase - FPLC fast protein liquid chromatography     

Enhancement of phenol biodegradation by Ochrobactrum sp. isolated from industrial wastewaters

Ochrobactrum sp., was tested with regard to its phenol degradation capacity at different pH levels, and with different carbon sources (mineral salt medium with glucose (MSG) and the same medium with 0.5%, 1%, and 2% (v/v) molasses (MSM)) and phenol concentrations. The highest degradation was in mineral salt medium with 1% (v/v) molasses (45.9%), while degradation was 21.1% in mineral salt medium with 5 g l⁻¹ glucose. These data show that the addition of molasses to mineral salt medium enhanced phenol degradation by Ochrobactrum sp. The bacterium can be used effectively to treat wastewaters containing phenol.     

Biodegradation and detoxification of nicotine in tobacco solid waste by a Pseudomonas sp.

A Pseudomonas sp. grew with nicotine optimally 3 g l^–1 and at 30 °C and pH 7. Nicotine was fully degraded within 10 h. The resting cells degraded nicotine in tobacco solid waste completely within 6 h in 0.02 m sodium phosphate buffer (pH 7) at maximally 56 mg nicotine h^–1 g dry cell^–1.     

Nocardia rhamnosiphila sp. nov., isolated from soil

In this study two actinomycete strains were isolated in Cape Town (South Africa), one from a compost heap (strain 202GMO^T) and the other from within the fynbos-rich area surrounded by the horseracing track at Kenilworth Racecourse (strain C2). Based on 16S rRNA gene sequence BLAST analysis, the strains were identified as members of the genus Nocardia. Phylogenetic analysis showed that the strains clustered together and are most closely related to Nocardia flavorosea NRRL B-16176^T, Nocardia testacea JCM 12235^T, Nocardia sienata IFM 10088^T and Nocardia carnea DSM 43397^T. This association was also supported by gyrB based phylogenetic analysis. The results of DNA–DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of both strains 202GMO^T and C2 from related species. However, their high DNA relatedness showed that they belong to the same species. Strain 202GMO^T was selected as the type strain to represent this novel species, for which the name Nocardia rhamnosiphila is proposed (=DSM 45147^T = NRRL B-24637^T).     

Biodegradation of exploded cotton stalk by Bacillus sp.

The exploded bast, branch and stem of cotton stalk were degraded by alkalophilic Bacillus NT-19, with weight losses of 24%, 20% and 14%, respectively, after 14 d. Compared with a white-rot fungus (Phanerochaete chrysosporium), Bacillus NT-19 preferentially degraded the non-cellulose components of cotton stem. The relative degree of crystallinity of bast fibers decreased by 8% and the middle lamella was partially removed from the fiber bundle by the Bacillus.     

Acid-precipitable polymeric lignin from hardwood lignocellulose: production by a Streptomyces sp.

A Streptomyces sp. isolate, from decayed wood shavings, solubilized lignocellulose (LC) and lignin of Pinus radiata, producing about 50 mg acid-precipitable polymeric lignin per g LC. The product was poor in protein and carbohydrates and contained mainly vanillin, guaicol, vanillic and ferulic acids. Hardwood LC is thus suitable for producing APPL as a phenolic chemical feedstock.V.M. Kaluskar is with the Department of Microbiology, J and J Science College, Nadiad 387001, Gujarat, India. B.P. Kapadanis is with the Department of Microbiology, School of Sciences, University of Pune, Ganesh Khind, Pune-41107, Maharashtra, India. M.J. Penninckx is with the Unit of Microbial Physiology and Ecology, Free University of Brussels, c/o IPB 642, rue Engeland, B-1180, Brussels, Belgium     

Decolorization of triphenylmethane and azo dyes by Citrobacter sp.

A Citrobacter sp., isolated from soil at an effluent treatment plant of a textile and dyeing industry, decolorized several recalcitrant dyes except Bromophenol Blue. More than 90% of Crystal Violet and Methyl Red at 100 M were reduced within 1 h. Gentian Violet, Malachite Green and Brilliant Green lost over 80% of their colors in the same condition, but the percentage decolorization of Basic Fuchsin and Congo Red were less than the others, 66 and 26%, respectively. Decolorization of Congo Red was mainly due to adsorption to cells. Color removal was optimal at pH 7–9 and 35–40 °C. Decolorization of dyes was also observed with extracellular culture filtrate, indicating the color removal by enzymatic biodegradation.     

Degradation of lignin and lignin model compounds by various mutants of the white-rot fungus Sporotrichum pulverulentum

In order to better understand which enzyme are of importance in lignin degradation, new cellulase deficient strains from Sporotrichum pulverulentum have been isolated by spontaneous and induced mutations from both wild type and from the earlier studied cellulase deficient strain 44. These new strains are xylanase positive (Xyl⁺), and produce considerably higher amounts of phenol oxidases (Pox) than either parent type. The new strains have been compared with the wild type and strain 44 with respect to their ability to release ¹⁴CO₂ from a) vanillic acid labelled in the carboxyl, methoxyl and ring carbons; b) the dimer (4-methoxy-¹⁴C)-veratryl-glycerol--guaiacyl ether; c) ¹⁴C-ring-labelled DHP and ¹⁴C[-carbon side chain] labelled DHP.The new strains, the wild type and strain 44 were compared with respect to their ability to cause weight losses in wood blocks and to delignify wood. One of the new strains, 63-2, caused a higher weight loss in wood than either the wild type or strain 44. Another strain, 44-2, produced a higher weight loss than strain 44. An increase in acid-soluble lignin was observed in wood blocks treated for two weeks with the two new mutant strains and wild type. After prolonged incubation for 6 and 8 weeks the amount of acid-soluble lignin decreased.Abbreviations DHP Dehydrogenation polymerizate - DMS 2,2-dimethylsuccinic acid     

Enzymatic characterization of Nocardia spp. and related bacteria by API ZYM profile

Characterization of 62 isolates belonging to the genus Nocardia and related bacteria was obtained by using the API ZYM system. The difference in enzymatic profile should allow a relatively efficient, low-cost means to identify aerobic actinomycetes of clinical significance.     

Nocardia lijiangensis sp. nov., a novel actinomycete strain isolated from soil in China

A novel actinomycete strain YIM 33378T was isolated from a soil sample collected from Lijiang, Yunnan Province, China. Based on the results of phenotypic and genotypic characteristics, strain YIM 33378T should be assigned to a new species of the genus Nocardia, for which the name Nocardia lijiangensis sp. nov. is proposed. The type strain is YIM 33378T (= CCTCC AA 204005T = KCTC 19028T). The GenBank accession number for the sequence reported in this paper is AY779043.     

Nocardia alba sp.nov., a novel actinomycete strain isolated from soil in China

A novel actinomycete strain, designated YIM 30243T, was isolated from a soil sample in Yunnan Province, China. Based on the results of phenotypic and genotypic characteristics, strain YIM 30243T should be assigned to a new species of the genus Nocardia, for which the name Nocardia alba sp. nov. is proposed. The type strain is YIM 30243T (= CCTCC AA001030T = DSM 44684T).     

Microbial degradation of tyre rubber particles

Degradation of rubber particles from tyre treads, having diameters from 0.8 to 2.3 mm, was achieved using Nocardia sp. 835A-Rc, a mutant strain with strong rubber-degrading ability. The entire surface of the particles was uniformly attacked by the organism either without stirring of the culture medium or at a very low stirring rate of 40 rpm. At a higher rate of stirring, however, a small number of large microbial colonies were formed on the rubber surface and separate deep semi-spherical cavities were observed after the removal of microbial cells by washing. The number of microbial colonies decreased with increasing stirring rate but each one of the colonies became larger at the same time. As the result of these two counteracting effects of stirring on microbial activity, the weight loss of the particles increased when the stirring rate was raised from 0 to 40 rpm but decreased when the rate was increased from 40 to 70 or 150 rpm. At the stirring rate of 40 rpm, the weight losses of the particles with mean diameters of about 0.8, 1.1 and 2.3 mm were 57, 50 and 36%, respectively, after 8 weeks. The rate of microbial degradation increased again when the stirring was raised from 150 to 300 rpm.     

The importance of phenol oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum

The lignin degradation abilities of wildtype, a phenol oxidase-less mutant and a phenol oxidase-positive revertant of Sporotrichum pulverulentum were compared to determine if phenol oxidase activity is necessary for lignin degradation by white-rot fungi. The phenol oxidase-less mutant was unable to degrade kraft lignin or wood. The phenol oxidase-positive revertant, however, regained the ability of the wildtype to degrade kraft lignin and all of the major components of wood. It was found that kraft lignin and lignin-related phenols decreased cellulase and xylanase production by the phenol oxidase-less mutant. Addition of highly purified laccase increased the production of endo-1,4--glucanase in the phenol oxidase-less mutant in the presence of vanillic acid and kraft lignin. After addition of laccase to kraft lignin agar plates, the phenol oxidase-less mutant could degrade kraft lignin.It is proposed that phenol oxidase function in regulating the production of both lignin-and polysaccharide-degrading enzymes by oxidation of lignin and lignin-related phenols when S. pulverulentum is growing on wood.Abbreviation WT wildtype Sporotrichum pulverulentum Research supported by a grant from Stiftelsen Nils and Dorthi Troëdssons forskningsfond     

Four novel yeasts from decaying organic matter: Blastobotrys robertii sp. nov., Candida cretensis sp. nov., Candida scorzettiae sp. nov. and Candida vadensis sp. nov

Four novel yeast species are described, two from decaying mushrooms, viz. Candida cretensis and Candida vadensis, and two from rotten wood, viz. Blastobotrys robertii and Candida scorzettiae. Accession numbers for the CBS and ARS Culture Collections, and GenBank accession numbers for the D1/D2 domains of the large subunit of ribosomal DNA are: B. robertii CBS 10106^T, NRRL Y-27775, DQ839395; C. cretensis CBS 9453^T, NRRL Y-27777, AY4998861 and DQ839393; C. scorzettiae CBS 10107^T, NRRL Y-27665, DQ839394; C. vadensis CBS 9454^T, NRRL Y-27778, AY498863 and DQ839396. The GenBank accession number for the ITS region of C. cretensis is AY498862 and that for C. vadensis is AY498864. C. cretensis was the only species of the four that displayed fermentative activity. All four type strains grew on n-hexadecane. C. scorzettiae is the only one of the new species that assimilates some phenolic compounds, viz. 3-hydroxy derivatives of benzoic, phenylacetic and cinnamic acids, but not the corresponding 4-hydroxy acids. This is indicative of an operative gentisate pathway.     

Degradation of lignin and lignin related compounds by protoplasts isolated from Fomes annosus

Protoplasts from a lignolytic fungus Fomes annosus were prepared through enzymatic hydrolysis of mycelium utilizing Novozym, a wall lytic enzyme preparation. Isolated protoplasts and living mycelium were compared in their ability to degrade ¹⁴C-labelled lignin related phenols and dehydropolymers of labelled coniferyl alcohol (synthetic lignin). The amounts of ¹⁴CO₂ released from O¹⁴CH₃-groups, ¹⁴C-2-side chains and ¹⁴C-rings by protoplasts was in the same range as those for intact mycelium. The methoxyl groups of synthetic lignin were more rapidly metabolized by protoplasts than by mycelium. When calculated in dpm of released ¹⁴CO₂ per mg protein the decomposition of ¹⁴C-labelled synthetic lignin and lignin-related monomers in a hyphae-free system of protoplasts was considerable higher than that obtained by the intact mycelium. The presence of intact hyphae is thus not necessary for lignin degradation to occur.Non-common-abbreviations used DHP Dehydropolymer of coniferyl alcohol - LS lignosulfonates prepared from DHP