Degradation of Kraft Indulin Lignin by Streptomyces viridosporus and Streptomyces badius

Crawford and collaborators have studied extensively the solubilization of lignocellulose by two Streptomyces species, S. badius and S. viridosporus. Using a condensed industrial lignin essentially devoid of carbohydrates, Indulin AT, as the sole source of carbon, similar results were obtained: (i) the growths of the bacteria were optimum at pH 7.5 to 8.5; (ii) yeast extract was a better source of nitrogen than NH(4)Cl; (iii) the products of the depolymerization of Indulin were soluble, acid-precipitable polymers. When d-glucose was added as a secondary carbon source, it was used preferentially and the production of acid-precipitable polymers began only after the complete depletion of the sugar. On the assumption that the degradation of Indulin was catalyzed by enzymes, proteins found in the culture media and soluble and insoluble intracellular proteins were incubated with Indulin at pH 7.0 at 37 degrees C. Proteins in all fractions from S. badius had ligninolytic activities which, with the exception of those in the intracellular soluble fraction, were increased in the presence of H(2)O(2). In S. viridosporus, both extra- and intracellular soluble activities were found which were not increased by H(2)O(2). The extracellular activity of S. viridosporus was not affected by heat, resisted partially an exposure to pH 1.0, and was completely destroyed by proteolysis.     

Mercury Resistance Is Encoded by Transferable Giant Linear Plasmids in Two Chesapeake Bay Streptomyces Strains

The Streptomyces strains CHR3 and CHR28, isolated from the Baltimore Inner Harbor, contained two and one, respectively, giant linear plasmids which carry terminally bound proteins. The plasmids pRJ3L (322 kb), from CHR3, and pRJ28 (330 kb), from CHR28, carry genes homologous to the previously characterized chromosomal Streptomyces lividans 66 operon encoding resistance against mercuric compounds. Both plasmids are transmissible (without any detectable rearrangement) to the chloramphenicol-resistant S. lividans TK24 strain lacking plasmids and carrying a chromosomal deletion of the mer operon. S. lividans TK24 conjugants harboring pRJ3L or pRJ28 exhibited profiles of mercury resistance to mercuric compounds similar to those of Streptomyces strains CHR3 and CHR28.     

Production and Characterization of Polymeric Lignin Degradation Intermediates from Two Different Streptomyces spp

Previous investigations have identified a quantitatively major intermediate of lignin degradation by Streptomyces viridosporus. The intermediate, a modified lignin polymer, acid-precipitable polymeric lignin (APPL), is released as a water-soluble catabolite and has been recovered in amounts equivalent to 30% of the lignin originally present in a corn stover lignocellulose substrate after degradation by this actinomycete. In the present work, APPLs were collected at various time intervals from cultures of two highly ligninolytic Streptomyces sp. strains, S. viridosporus T7A and S. badius 252, growing on corn stover lignocellulose. APPL production was measured over time, and the chemistry of APPLs produced by each organism after different time intervals was compared. Chemical characterizations included assays for lignin, carbohydrate, and ash contents, molecular weight distributions by gel permeation chromatography, and chemical degradation analyses by permanganate oxidation, acidolysis, and alkaline ester hydrolysis. Differences between the organisms were observed in the cultural conditions required for APPL production and in the time courses of APPL accumulation. S. viridosporus produced APPL in solid-state fermentation over a 6- to 8-week incubation period, whereas S. badius produced as much or more APPL, but only in liquid culture and over a 7- to 8-day incubation period. The chemistry of the APPLs produced also differed. S. viridosporus APPL was more lignin-like than that of S. badius and was slowly modified further over time, although no change in molecular weight distribution over time was observed. In contrast, S. badius APPL was less lignin-like and increased substantially in average molecular weight over time. Results indicated that differing mechanisms of lignin metabolism may exist in these two Streptomyces sp. strains. S. viridosporus APPL probably originates from the heart of the lignin and is released largely as the result of beta-ether cleavage and other oxidative reactions. S. badius APPL probably originates in the same manner; however, after release as a water-soluble catabolite, lower-molecular-weight intermediates of lignin degradation are repolymerized with APPL in a reaction catalyzed by an extracellular phenol oxidase. The chemical analyses and the presence of extracellular phenol oxidase in S. badius, but not in S. viridosporus, support this conclusion.     

Extracellular Enzyme Activities during Lignocellulose Degradation by Streptomyces spp.: A Comparative Study of Wild-Type and Genetically Manipulated Strains

The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Isoenzymes were analyzed by polyacrylamide gel electrophoresis and activity staining on the gels. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed.     

Paper Mill Effluent Decolorization by Fifty Streptomyces Strains

Fifty actinomycete strains isolated from lignocellulosic substrates were examined for the ability to remove the color from a paper mill effluent obtained after semichemical alkaline pulping of wheat straw. Streptomyces sp. strains UAH 15, UAH 23, UAH 30, and UAH 51 were selected for their ability to decolorize the effluent in a liquid medium containing 1% (wt/vol) glycerol, 0.2% (wt/vol) ammonium sulfate, and 80% (vol/vol) effluent. The highest levels of decolorization achieved after the strains grew were 60 to 65%. Strains UAH 30 and UAH 51 were selected for further study because of their different patterns of effluent decolorization during growth. Fractionation of the decolorized effluent by gel permeation chromatography demonstrated that there were reductions in the levels of absorbance of the high- and medium-molecular-weight compounds. These fractions were mainly responsible for the color of the effluent, while the last fractions, the low-molecular-weight compounds, could have been responsible for the residual color of the decolorized effluent. Thin-layer chromatography revealed significant differences among the patterns of bands corresponding to the acidified supernatants obtained after precipitation of alkali-lignin from the effluent samples decolorized by different Streptomyces strains.     

Degradation of polychlorinated phenols by Streptomyces rochei 303

The strain Streptomyces rochei 303 (VKM Ac-1284D) is capable of utilizing 2-chloro-,2,4-,2,6-dichloro- and 2,4,6-trichlorophenols as the sole source of carbon. Its resting cells completely dechlorinated and degraded 2-, 3-chloro-; 2,4-, 2,6-, 2,3-, 2,5-, 3,4-, 3,5-dichloro-; 2,4-, 2,6-dibromo-; 2,4,6-, 2,4,5-, 2,3,4-, 2,3,5-, 2,3,6-trichlorophenols; 2,3,5,6-tetrachloro- and pentachlorophenol. During chlorophenol degradation, a stoichiometric amount of chloride ions was released and chlorohydroquinols were formed as intermediates. In cell-free extracts of S. rochei, the activity of hydroxyquinol 1,2-dioxygenase was found. The enzyme was induced with chlorophenols. Of all so far described strains degrading polychlorophenols, S. rochei 303 utilized a wider range of chlorinated phenols as the sole sourse of carbon and energy.Abbreviations CP chlorophenol - DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol - DBrP dibromophenol - CHQ chlorohydroquinol - DCHQ dichlorohydroquinol - HHQ hydroxyhydroquinol - CHHQ chlorohydroxyhydroquinol - CC chlorocatechol - TLC thin layer chromatography - GC/MC chromato-mass-spectrometry - HPLC high-performance liquid chromatography     

Production and Characterization of Polymeric Lignin Degradation Intermediates from Two Different Streptomyces spp.

Previous investigations have identified a quantitatively major intermediate of lignin degradation by Streptomyces viridosporus. The intermediate, a modified lignin polymer, acid-precipitable polymeric lignin (APPL), is released as a water-soluble catabolite and has been recovered in amounts equivalent to 30% of the lignin originally present in a corn stover lignocellulose substrate after degradation by this actinomycete. In the present work, APPLs were collected at various time intervals from cultures of two highly ligninolytic Streptomyces sp. strains, S. viridosporus T7A and S. badius 252, growing on corn stover lignocellulose. APPL production was measured over time, and the chemistry of APPLs produced by each organism after different time intervals was compared. Chemical characterizations included assays for lignin, carbohydrate, and ash contents, molecular weight distributions by gel permeation chromatography, and chemical degradation analyses by permanganate oxidation, acidolysis, and alkaline ester hydrolysis. Differences between the organisms were observed in the cultural conditions required for APPL production and in the time courses of APPL accumulation. S. viridosporus produced APPL in solid-state fermentation over a 6- to 8-week incubation period, whereas S. badius produced as much or more APPL, but only in liquid culture and over a 7- to 8-day incubation period. The chemistry of the APPLs produced also differed. S. viridosporus APPL was more lignin-like than that of S. badius and was slowly modified further over time, although no change in molecular weight distribution over time was observed. In contrast, S. badius APPL was less lignin-like and increased substantially in average molecular weight over time. Results indicated that differing mechanisms of lignin metabolism may exist in these two Streptomyces sp. strains. S. viridosporus APPL probably originates from the heart of the lignin and is released largely as the result of β-ether cleavage and other oxidative reactions. S. badius APPL probably originates in the same manner; however, after release as a water-soluble catabolite, lower-molecular-weight intermediates of lignin degradation are repolymerized with APPL in a reaction catalyzed by an extracellular phenol oxidase. The chemical analyses and the presence of extracellular phenol oxidase in S. badius, but not in S. viridosporus, support this conclusion.     

Degradation of Bermuda and Orchard Grass by Species of Ruminal Bacteria

Fiber degradation in Bermuda grass and orchard grass was evaluated gravimetrically and by scanning and transmission electron microscopy after incubation with pure cultures of rumen bacteria. Lachnospira multiparus D-32 was unable to degrade plant cell wall components. Butyrivibrio fibrisolvens 49 degraded 6 and 14.9% of the fiber components in Bermuda grass and orchard grass, respectively, and Ruminococcus albus 7 degraded 11.4% orchard grass fiber but none in Bermuda grass. Both B. fibrisolvens and R. albus lacked capsules, did not adhere to fiber, and degraded only portions of the more easily available plant cell walls. R. flavefaciens FD-1 was the most active fiber digester, degrading 8.2 and 55.3% of Bermuda and orchard grass fiber, respectively. The microbe had a distinct capsule and adhered to fiber, especially that which is slowly degraded, but was able to cause erosion and disorganization of the more easily digested cell walls, apparently by extracellular enzymes. Results indicated that more digestible cell walls could be partially degraded by enzymes disassociated from cellulolytic and noncellulolytic bacteria, and data were consistent with the hypothesis that the more slowly degraded plant walls required attachment. Microbial species as well as the cell wall architecture influenced the physical association with and digestion of plant fiber.     

Degradation of 1,2,4-Trichloro- and 1,2,4,5-Tetrachlorobenzene by Pseudomonas Strains

Two Pseudomonas sp. strains, capable of growth on chlorinated benzenes as the sole source of carbon and energy, were isolated by selective enrichment from soil samples of an industrial waste deposit. Strain PS12 grew on monochlorobenzene, all three isomeric dichlorobenzenes, and 1,2,4-trichlorobenzene (1,2,4-TCB). Strain PS14 additionally used 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB). During growth on these compounds both strains released stoichiometric amounts of chloride ions. The first steps of the catabolism of 1,2,4-TCB and 1,2,4,5-TeCB proceeded via dioxygenation of the aromatic nuclei and furnished 3,4,6-trichlorocatechol. The intermediary cis-3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene (TCB dihydrodiol) formed from 1,2,4-TCB was rearomatized by an NAD⁺-dependent dihydrodiol dehydrogenase activity, while in the case of 1,2,4,5-TeCB oxidation the catechol was obviously produced by spontaneous elimination of hydrogen chloride from the initially formed 1,3,4,6-tetrachloro-1,2-dihydroxycyclohexa-3,5-diene. Subsequent ortho cleavage was catalyzed by a type II catechol 1,2-dioxygenase producing the corresponding 2,3,5-trichloromuconate which was channeled into the tricarboxylic acid pathway via an ordinary degradation sequence, which in the present case included 2-chloro-3-oxoadipate. From the structure-related compound 2,4,5-trichloronitrobenzene the nitro group was released as nitrite, leaving the above metabolite as 3,4,6-trichlorocatechol. Enzyme activities for the oxidation of chlorobenzenes and halogenated metabolites were induced by both strains during growth on these haloaromatics and, to a considerable extent, during growth of strain PS12 on acetate.     

Preparation, Characterization, and Microbial Degradation of Specifically Radiolabeled [14C]Lignocelluloses from Marine and Freshwater Macrophytes

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [¹⁴C]phenylalanine, [¹⁴C]tyrosine, and [¹⁴C]cinnamic acid as precursors. Specifically radiolabeled [¹⁴C-polysaccharide]lignocelluloses were prepared by using [¹⁴C]glucose as precursor. The rates of microbial degradation varied among [¹⁴C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [¹⁴C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [¹⁴C]phenylalanine and [¹⁴C]tyrosine were found associated with protein, although very little (3%) radioactivity from [¹⁴C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [¹⁴C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [¹⁴C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to ¹⁴CO₂; during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.     

Two New Mycobacterium Strains and Their Role in Toluene Degradation in a Contaminated Stream

Two toluene-degrading strains, T103 and T104, were isolated from rock surface biomass in a freshwater stream contaminated with toluene. The strains exhibit different capacities for degradation of toluene and other aromatic compounds and have characteristics of the genus Mycobacterium. Both are aerobic, rod-shaped, gram-positive, nonmotile, and acid-alcohol fast and produce yellow pigments. They have mainly straight-chain saturated and monounsaturated fatty acids with 10 to 20 carbon atoms and large amounts of tuberculostearic acid that are typical of mycobacteria. Fatty acid analyses indicate that T103 and T104 are different mycobacterial strains that are related at the subspecies level. Their identical 16S rDNA sequences are most similar to Mycobacterium aurum and Mycobacterium komossense, and they constitute a new species of fast-growing mycobacteria. Ecological studies reveal that toluene contamination has enriched for toluene-degrading bacteria in the epilithic microbial community. Strains T103 and T104 play only a small role in toluene degradation in the stream, although they are present in the habitat and can degrade toluene. Other microorganisms are consequently implicated in the biodegradation.     

Enzymatic Phosphorylation of Streptomycin by Extracts of Streptomycin-producing Strains of Streptomyces

Extracts of post-exponential phase mycelia of Streptomyces bikiniensis ATCC 11062, and other streptomycin-producers, catalyze phosphorylation of streptomycin and dihydrostreptomycin with adenosine-5'-triphosphate. The phosphate is esterified with an -OH group of the streptidine moiety. It is suggested that O-phosphoryl-streptomycin might serve as an intracellular precursor of extracellular streptomycin or as a detoxification product of streptomycin or that it might serve an unknown physiological function in the producing organism.     

Degradation of Phenanthrene by Mutant Naphthalene-Degrading Pseudomonas putida Strains

Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-born genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.     

Effects of pH on Lignin and Cellulose Degradation by Streptomyces viridosporus

Lignocellulose degradation by Streptomyces viridosporus results in the oxidative depolymerization of lignin and the production of a water-soluble lignin polymer, acid-precipitable polymeric lignin (APPL). The effects of the culture pH on lignin and cellulose metabolism and APPL production by S. viridosporus are reported. Dry, ground, hot-water-extracted corn (Zea mays) lignocellulose was autoclaved in 1-liter reagent bottles (5 g per bottle) and inoculated with 50-ml volumes of S. viridosporus cells suspended in buffers of specific pH (pH 6.0 to 9.2 at 0.4 pH unit intervals). Four replicates of inoculated cultures and of uninoculated controls at each pH were incubated as solid-state fermentations at 37°C. After 6 weeks of incubation the percent loss of lignocellulose, lignin, and carbohydrate and the amount of APPL produced were determined for each replicate. Optimal lignocellulose degradation, as shown by substrate weight loss, was observed in the pH range of 8.4 to 8.8. Only minor differences were seen in the Klason lignin, carbohydrate, protein, and ash contents of the APPLS produced by cultures at each pH. The effects of pH on the degradation of a spruce (Picea pungens) [¹⁴C-lignin]lignocellulose and a Douglas fir (Pseudotsuga menziesii) [¹⁴C-glucan]-lignocellulose were also determined at pH values between 6.5 and 9.5 (0.5 pH unit intervals). The incubations were carried out for 3 weeks at 37°C with bubbler-tube cultures. The percentage of initial ¹⁴C recovered as ¹⁴CO₂, ¹⁴C-labeled water-soluble products, and [¹⁴C]APPL was then determined. The mineralization of lignin and cellulose to CO₂ was optimal at pHs 6.5 and 7.0, respectively. However, the optimum for lignin and cellulose solubilization was pH 8.5, which correlated with the pH 8.5 optimum for APPL production. Overall, the data show that, whereas lignin mineralization is optimal at neutral to slightly acidic pHs, lignocellulose degradation with lignin solubilization and APPL production is promoted by alkaline pHs. These findings indicate that lignin-solubilizing actinomycetes may play an important role in the metabolism of lignin in neutral to alkaline soils in which ligninolytic fungi are not highly competitive.     

Lignin Degradation by Streptomyces viridosporus: Isolation and Characterization of a New Polymeric Lignin Degradation Intermediate

A new, quantitatively significant intermediate formed during lignin degradation by Streptomyces viridosporus T7A was isolated and characterized. In Streptomyces-inoculated cultures, the intermediate, an acid-precipitable, polyphenolic, polymeric lignin (APPL), accumulated in the growth medium. The APPL was a water-soluble polymer probably consisting of a heterogeneous mixture of molecular weight components of ≥20,000. APPLs were precipitable from culture filtrates after they had been acidified to pH <3 to 5. Noninoculated controls yielded little APPL, but supernatant solutions from inoculated cultures produced quantities of APPL that correlated with the biodegradability of the lignocellulose type. Maximal recovery of APPL was obtained from corn lignocellulose, reaching 30% of the initial lignin present in the substrate. APPLs contained small amounts of carbohydrate, organic nitrogen, and inorganic materials. The lignin origin of APPLs was confirmed by chemical analyses, which included acidolysis, permanganate oxidation, elemental analyses, functional group analyses, nuclear magnetic resonance spectroscopy, and ¹⁴C isotopic techniques. Analyses of APPLs from corn lignocelluloses showed that S. viridosporus-degraded APPLs were lignin derived but significantly different in structure from APPLs derived from uninoculated controls or from a standard corn milled-wood lignin. Degraded APPLs were enriched in phenolic hydroxyl groups and, to a small extent, in carboxyl groups. Degradative changes appeared to be largely oxidative and were thought to involve substantial cleavage of p-hydroxy ether linkages and methoxyl groups in the lignin.