Characterization oftdtgenes for the degradation of tricyclic diterpenes byPseudomonas diterpeniphilaA19-6a

2002 ◽  
Vol 48 (1) ◽  
pp. 49-59 ◽  
Author(s):  
C A Morgan ◽  
R C Wyndham
Keyword(s):  
Gene Knockout ◽  
Resin Acid ◽  
Pulp Mill ◽  
Ring Structure ◽  
Resin Acids ◽  
P450 Monooxygenase ◽  
Aquatic Life ◽  
Degrading Bacterium ◽  
Coa Ligase ◽  
Pulp Mill Effluents

Resin acids are tricyclic diterpenes that are toxic to aquatic life when released in high concentrations in pulp mill effluents. These naturally formed organic acids are readily degraded by bacteria and fungi; nevertheless, many of the mechanisms involved are still unknown. We report the localization, cloning, and sequencing of genes for abietane degradation (9.18 kb; designated tdt (tricyclic diterpene) LRSABCD) from the γ-Proteobacterium Pseudomonas diterpeniphila A19-6a. Using gene knockout mutants, we demonstrate that tdtL, encoding a putative CoA ligase, is required for growth on abietic and dehydroabietic acids. A second gene knockout in tdtD, encoding a putative cytochrome P450 monooxygenase, reduced the growth of strain A19-6a on abietic and dehydroabietic acids as sole sources of carbon and energy, but did not eliminate growth. The degree of homology between P450TdtDand P450TerpC, the closest known P450 homologue to TdtD, identifies TdtD as a new member of the P450 superfamily. Hybridization of six of the tdt genes to genomic DNA of a related resin acid degrading bacterium Pseudomonas abietaniphila BKME-9 identified tdt homologues in this strain that utilizes aromatic ring dioxygenase genes (dit) to open the ring structure of abietic and dehydroabietic acids. These results suggest the tdt and dit genes may function in concert to allow these Pseudomonas strains to degrade resin acids. Homologues of several of the tdt genes were detected in resin acid degrading Ralstonia and Comamonas species within the β- and γ-Proteobacteria.Key words: resin acid, tdt gene, biodegradation, Pseudomonas.

Microbiology and biodegradation of resin acids in pulp mill effluents: a minireview

1997 ◽  
Vol 43 (7) ◽  
pp. 599-611 ◽  
Author(s):  
Steven N. Liss ◽  
Paul A. Bicho ◽  
John N. Saddler
Keyword(s):  
Biological Treatment ◽  
Resin Acid ◽  
Pulp Mill ◽  
Paper Mill ◽  
Transformation Products ◽  
Conclusive Evidence ◽  
Resin Acids ◽  
Original Material ◽  
Pulp And Paper Mill ◽  
Pulp Mill Effluents

Resin acids, a group of diterpenoid carboxylic acids present mainly in softwood species, are present in many pulp mill effluents and toxic to fish in recipient waters. They are considered to be readily biodegradable. However, their removal across biological treatment systems has been shown to vary. Recent studies indicate that natural resin acids and transformation products may accumulate in sediments and pose acute and chronic toxicity to fish. Several resin acid biotransformation compounds have also been shown to bioaccumulate and to be more resistant to biodegradation than the original material. Until recently, the microbiology of resin-acid degradation has received only scant attention. Although wood-inhabiting fungi have been shown to decrease the level of resin present in wood, there is no conclusive evidence that fungi can completely degrade these compounds. In contrast, a number of bacterial isolates have recently been described which are able to utilize dehydroabietic or isopimaric acids as their sole carbon source. There appears to be an unusually high degree of substrate specificity with respect to the utilization of abietane congeners and the presence of substituents. Pimaranes do not appear to be attacked to the same extent as the abietanes. This paper reviews the occurrence, chemistry, toxicity, and biodegradation of resin acids in relation to the biological treatment of pulp and paper mill effluents.Key words: resin acids, biodegradation, pulp mill effluents.

Resin acid degradation by bacterial strains grown on CTMP effluent

1997 ◽  
Vol 35 (2-3) ◽  
pp. 33-39
Author(s):  
Yi Zhang ◽  
P. A. Bicho ◽  
C. Breuil ◽  
J. N. Saddler ◽  
S. N. Liss
Keyword(s):  
Biological Treatment ◽  
Resin Acid ◽  
Pulp Mill ◽  
Dehydroabietic Acid ◽  
Operating Conditions ◽  
Resin Acids ◽  
Organic Substrates ◽  
Bacterial Strains ◽  
Pulp Mill Effluents ◽  
Chemithermomechanical Pulping

All resin acids are diterpenoid carboxylic acids that are components of softwood extractives and they are known to contribute to much of the toxicity of pulp mill effluents. Although biological treatment systems can efficiently remove resin acids during normal operating conditions, resin acid breakthroughs occasionally occur. Recently we isolated five bacterial strains from bleach kraft effluents that degrade dehydroabietic acid (DHA), a resin acid commonly found in effluents. In this study we examined the ability of two bacterial strains (BKME 5 and BKME 9) to grow on chemithermomechanical pulping (CTMP) effluent and degrade DHA. Both of the strains could grow on CTMP effluents, but did not degrade DHA. COD measurement showed that both strains used other organic substrates in CTMP effluent. When nutrients (NH4⊕, PO43−, minerals and vitamins) were added to the effluent, both growth and DHA degradation increased significantly. The strains used DHA and other organic sources in the CTMP effluent simultaneously. The stimulated growth resulting from use of other organic material did not increase the rate of DHA degradation. It was found that ammonium played an important role in the DHA degradation of both strains. Without added ammonium, DHA degradation did not occur. Other nutrients also played important roles in DHA degradation by BKME 9.

Development of an Immunoassay for the Detection of Resin Acids in Pulp Mill Effluents

1995 ◽  
pp. 675-688
Author(s):  
Kai Li ◽  
Michael Chester ◽  
James P. Kutney ◽  
Colette Breuil ◽  
John N. Saddler
Keyword(s):  
Pulp Mill ◽  
Resin Acids ◽  
Pulp Mill Effluents

Isolation and characterization of thermophilic bacteria capable of degrading dehydroabietic acid

1999 ◽  
Vol 45 (6) ◽  
pp. 513-519 ◽  
Author(s):  
Zhongtang Yu ◽  
William W Mohn
Keyword(s):  
Thermophilic Bacteria ◽  
Resin Acid ◽  
Pulp Mill ◽  
Maximum Growth ◽  
Dehydroabietic Acid ◽  
Resin Acids ◽  
Bacterial Strains ◽  
Rdna Sequences ◽  
Isolation And Characterization ◽  
Continuous Enrichment

Using a semi-continuous enrichment method, we isolated two thermophilic bacterial strains, which could completely degrade abietane resin acids, including dehydroabietic acid (DhA). Strain DhA-73, isolated from a laboratory-scale bioreactor treating bleached kraft mill effluent at 55°C, grew on DhA as sole carbon source; while DhA-71, isolated from municipal compost, required dilute tryptic soy broth for growth on DhA. DhA-71 grew on DhA from 30°C to 60°C with maximum growth at 50°C; while, DhA-73 grew on DhA from 37°C to 60°C with maximum growth at 55°C. At 55°C, the doubling times for DhA-71 and DhA-73 were 3.3 and 3.7 h, respectively. DhA-71 and DhA-73 had growth yields of 0.26 and 0.19 g of protein per g of DhA, respectively. During growth on DhA, both strains converted DhA to CO2, biomass, and dissolved organic carbon. Analyses of the 16S-rDNA sequences of these two strains suggest that they belong to two new genera in theRubrivivax subgroup of the beta subclass of the Proteobacteria. Strains DhA-71 and DhA-73 are the first two bacteria isolated and characterized that are capable of biodegradation of resin acids at high temperatures. This study provided direct evidence for biodegradation of resin acids and feasibility for biotreatment of pulp mill effluent at elevated temperatures.Key words: biodegradation, resin acid, semi-continuous enrichment, thermophiles.

scholarly journals Monitoring Gene Expression in Mixed Microbial Communities by Using DNA Microarrays

2003 ◽  
Vol 69 (2) ◽  
pp. 769-778 ◽  
Author(s):  
Philip Dennis ◽  
Elizabeth A. Edwards ◽  
Steven N. Liss ◽  
Roberta Fulthorpe
Keyword(s):  
Gene Expression ◽  
Microbial Communities ◽  
Ralstonia Eutropha ◽  
Batch Reactor ◽  
Resin Acid ◽  
Pulp Mill ◽  
Dehydroabietic Acid ◽  
Bacterial Gene ◽  
Degrading Bacterium ◽  
Catabolic Genes

ABSTRACT A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 105 cells/ml was clearly detected against a background of 108 cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 106 cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.

Biochemistry and Ecology of Resin Acid Biodegradation in Pulp and Paper Mill Effluent Treatment Systems

1999 ◽  
Vol 40 (11-12) ◽  
pp. 273-280 ◽  
Author(s):  
William W. Mohn ◽  
Vincent J. J. Martin ◽  
Zhongtang Yu
Keyword(s):  
Quantitative Pcr ◽  
Resin Acid ◽  
Pulp Mill ◽  
Paper Mill ◽  
Pulp And Paper ◽  
Resin Acids ◽  
Effluent Treatment ◽  
Biochemical Pathway ◽  
Pulp And Paper Mill ◽  
Paper Mill Effluents

A better understanding of the mechanisms and ecology of resin acid biodegradation will contribute to improved performance of existing treatment systems and development of new treatment systems for pulp and paper mill effluents. Using molecular genetic methods, we have partially elucidated the biochemical pathway for degradation of abietane resin acids by Pseudomonas abietaniphila BKME-9. We identified genes encoding putative membrane-associated proteins that are required for abietane metabolism. These proteins may function in cellular uptake of, or response to, resin acids. Genetic and physiological evidence suggests that a monooxygenase is involved in the biochemical pathway. A quantitative PCR assay was developed for ditA1, a gene from BKME-9 encoding resin acid degradation. In an aerated lagoon treating pulp mill effluent, a population carrying ditA1 was found, which was a small fraction (10−7) of the total microbial community. This population was evenly distributed throughout the system and was a stable member of the community over time. Quantitative PCR assays were used to monitor Pseudomonas abietaniphila BKME-9 and Zoogloea resiniphila DhA-35 when they were separately used to inoculate a complex microbial communities in laboratory sequencing batch reactors. Both inocula were stably maintained in the community for 24 days. These inocula stimulated resin acid removal by the community when it was stressed by high pH or by high resin acid loading.

Microbiological Detoxificaion of Resin Acids

1986 ◽  
Vol 21 (1) ◽  
pp. 119-129 ◽  
Author(s):  
James A. Servizi ◽  
Dennis W. Martens ◽  
Robert W. Gordon ◽  
James P. Kutney ◽  
Mahatam Singh ◽  
...  
Keyword(s):  
Acute Toxicity ◽  
Daphnia Pulex ◽  
Pulp Mill ◽  
Initial Step ◽  
Resin Acids ◽  
Ring Cleavage ◽  
Mortierella Isabellina ◽  
Fold Reduction ◽  
Pulp Mill Effluents ◽  
Chlorine Removal

Abstract The fungus, Mortierella isabellina, was used to define the biochemical transformations required to detoxify three resin acids and three chlorinated resin acids commonly found in pulp mill effluents. Detoxification was based on bioassays using Daphnia pulex and, whenever feasible, sockeye fry (Oncorhynchus nerka). Hydroxylation at C(2a) generally was the initial step in detoxification and usually resulted in at least a ten-fold reduction in acute toxicity. Additional hydroxylation further reduced acute toxicity. Ring cleavage was not required to detoxify the resin acids or their chlorinated derivatives. Furthermore, substantial reductions in acute toxicities of chlorinated compounds were achieved without chlorine removal.

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