Enzyme recruitment allows the biodegradation of recalcitrant branched hydrocarbons by Pseudomonas citronellolis.

Experiments were carried out to construct pseudomonad strains capable of the biodegradation of certain recalcitrant branched hydrocarbons via a combination of alkane and citronellol degradative pathways. To promote the metabolism of the recalcitrant hydrocarbon 2,6-dimethyl-2-octene we transferred the OCT plasmid to Pseudomonas citronellolis, a pseudomonad containing the citronellol pathway. This extended the n-alkane substrate range of the organism, but did not permit utilization of the branched hydrocarbon even in the presence of a gratuitous inducer of the OCT plasmid. In a separate approach n-decane-utilizing (Dec+) mutants of P. citronellolis were selected and found to be constitutive for the expression of medium- to long-chain alkane oxidation. The Dec+ mutants were capable of degradation of 2,6-dimethyl-2-octene via the citronellol pathway as shown by (i) conversion of the hydrocarbon to citronellol, determined by gas-liquid chromatography-mass spectrometry, (ii) induction of geranyl-coenzyme A carboxylase, a key enzyme of the citronellol pathway, and (iii) demonstration of beta-decarboxymethylation of the hydrocarbon by whole cells. The Dec+ mutants had also acquired the capacity to metabolize other recalcitrant branched hydrocarbons such as 3,6-dimethyloctane and 2,6-dimethyldecane. These studies demonstrate how enzyme recruitment can provide a pathway for the biodegradation of otherwise recalcitrant branched hydrocarbons.     

A kinetic model for surfactant inhibition of pentachlorophenol biodegradation

A kinetic model is used to describe the effect of the nonionic surfactant Tergitol NP-10 (TNP10) on pentachlorophenol (PCP) biodegradation by Sphingomonas chlorophenolica sp. strain RA2. Different initial biomass to initial substrate ratios ranging from 13 to 418 were tested with 23 TNP10 concentrations ranging from 0 to 1500 mg/L. Tests were also conducted at 10 degrees C and 20 degrees C. No PCP biodegradation inhibition was observed at concentrations below the critical micelle concentration (CMC) of 50 mg/L. TNP10 concentrations above 100 to 200 mg/L were increasingly inhibitory to PCP biodegradation rates. This inhibition was best described by the Monod kinetic equation wherein the effect of TNP10 inhibition is reflected in the half-saturation constant (Ks). The value of the Ks increased from between 1.5 and 13.5 mg/L with no surfactant present to 44 to 131 mg/L at 1000 mg/L TNP10. Using a standard competitive inhibition approach, the inhibition constant for TNP10 was approximately 100 mg/L at both 10 degrees C and 20 degrees C.     

Syntaxin 2 and SNAP-23 are required for regulated surfactant secretion

The secretion of lung surfactant in alveolar type II cells is a complex process involving the fusion of lamellar bodies with the plasma membrane. This process is somewhat different from the exocytosis of hormones and neurotransmitters. For example, it is a relatively slower process, and lamellar bodies are very large vesicles with a diameter of approximately 1 microm. SNARE proteins are the conserved molecular machinery of exocytosis in the majority of secretory cells. However, their involvement in surfactant secretion has not been reported. Here, we showed that syntaxin 2 and SNAP-23 are expressed in alveolar type II cells. Both proteins are associated with the plasma membrane, and to some degree with lamellar bodies. An antisense oligonucleotide complementary to syntaxin 2 decreased its mRNA and protein levels. The same oligonucleotide also inhibited surfactant secretion, independent of secretagogues. A peptide derived from the N-terminus of syntaxin 2 or the C-terminus of SNAP-23 significantly inhibited Ca(2+)- and GTPgammaS-stimulated surfactant secretion from permeabilized type II cells in a dose-dependent manner. Furthermore, introduction of anti-syntaxin 2 or anti-SNAP-23 antibodies into permeabilized type II cells also inhibited surfactant release. Our results suggest that syntaxin 2 and SNAP-23 are required for regulated surfactant secretion.     

Metabolic pathway for the biodegradation of sodium dodecyl sulfate by Pseudomonas sp. C12B

Metabolism of sodium dodecyl sulfate (SDS) by the detergent-degrading bacterium Pseudomonas C12B has been studied using a 14C radiotracer in combination with radio-respirometry, radio-TLC, and GLC. Metabolism was extensive with 70% of the radiolabel released as 14CO2 at completion. The remainder of the radiolabel was incorporated almost totally into cells. Ether extraction of cells indicated that 14C-labeled cellular material appearing early in the uptake process was predominantly ether-extractable (mainly 1-dodecanol) and was subsequently converted to more polar metabolites. Analysis of the extractable lipids established the sequential production from [1-14C]SDS of 1-dodecanol, dodecanal, and dodecanoic acid. At this point the pathway diverged leading either to formation of 14CO2 via beta-oxidation or to elongation to C14, C16, and C18 fatty acyl residues with rapid incorporation into lipid fractions such as phospholipids. The pathway was correlated with known long-chain alkylsulfatases and alcohol dehydrogenases in this isolate and indicated that hydrophobic metabolites of the alkyl chain of surfactants can be incorporated into cellular components such as membrane lipids without prior degradation by beta-oxidation.     

Enzyme recruitment allows the biodegradation of recalcitrant branched hydrocarbons by Pseudomonas citronellolis

Experiments were carried out to construct pseudomonad strains capable of the biodegradation of certain recalcitrant branched hydrocarbons via a combination of alkane and citronellol degradative pathways. To promote the metabolism of the recalcitrant hydrocarbon 2,6-dimethyl-2-octene we transferred the OCT plasmid to Pseudomonas citronellolis, a pseudomonad containing the citronellol pathway. This extended the n-alkane substrate range of the organism, but did not permit utilization of the branched hydrocarbon even in the presence of a gratuitous inducer of the OCT plasmid. In a separate approach n-decane-utilizing (Dec+) mutants of P. citronellolis were selected and found to be constitutive for the expression of medium- to long-chain alkane oxidation. The Dec+ mutants were capable of degradation of 2,6-dimethyl-2-octene via the citronellol pathway as shown by (i) conversion of the hydrocarbon to citronellol, determined by gas-liquid chromatography-mass spectrometry, (ii) induction of geranyl-coenzyme A carboxylase, a key enzyme of the citronellol pathway, and (iii) demonstration of beta-decarboxymethylation of the hydrocarbon by whole cells. The Dec+ mutants had also acquired the capacity to metabolize other recalcitrant branched hydrocarbons such as 3,6-dimethyloctane and 2,6-dimethyldecane. These studies demonstrate how enzyme recruitment can provide a pathway for the biodegradation of otherwise recalcitrant branched hydrocarbons.     

Flocculation of Pseudomonaswith aluminum sulfate for enhanced biodegradation of synthetic dyes

A Pseudomonas strain, which could degrade synthetic azo dyes, was flocculated with aluminum sulfate (alum) for enforced immobilization. Alum at 800 mg/L was required to immobilize 60% of the cells in a nutrient-rich culture medium which contained 180 mg dry cell/L. The flocculation efficacy was consistent (83-85%) in a pH range from 4 to 10. Up to 95% of bacterial activity was lost when the cells were flocculated at a pH below 7 compared to about 70% activity lost at pH 8.     

Dual substrate biodegradation of a nonionic surfactant and pentachlorophenol by Sphingomonas chlorophenolica RA2

The simultaneous biodegradation of the nonionic surfactant Tween 20 (Tw20) and pentachlorophenol (PCP) by Sphingomonas chlorophenolica sp. Strain RA2 (RA2) was measured. As a sole substrate, Tw20 biodegradation was best described by the Contois kinetic model. During concurrent biodegradation of Tw20 and PCP, the biodegradation rates of Tw20 were not significantly affected by 50 or 100 mg/L PCP, but were significantly inhibited by 500 mg/L PCP. Decreases in cell yield in the presence of PCP suggest that PCP was acting as an uncoupler. Cultures were pre-grown on PCP or Tw20 before degradation of PCP to evaluate enzyme induction effects, and long lags before PCP biodegradation after growth on Tw20 occurred. Although biokinetic models could accurately describe some of the data sets of RA2 growth and Tw20 and PCP degradation, finding a single set of kinetic parameters that predicted all dual substrate tests was not achieved. The complicating factors to modeling PCP and Tw20 interactions are described and may be more widely applicable to the biodegradation of toxic organic compounds in the presence of a biodegradable surfactant.     

Cloning and sequencing of Pseudomonas genes determining sodium dodecyl sulfate biodegradation.

The nucleotide sequences of two genes involved in sodium dodecyl sulfate (SDS) degradation, by Pseudomonas, have been determined. One of these, sdsA, codes for an alkyl sulfatase (58,957 Da) and has similarity (31.8% identity over a 201-amino acid stretch) to the N terminus of a predicted protein of unknown function from Mycobacterium tuberculosis. The other gene, sdsB, codes for a positive activator protein (33,600 Da) that has extensive similarity with the lysR family of helix-turn-helix DNA-binding activator proteins.     

On the origin of the branched alkyl substituents on ring B of flexirubin-type pigments

Studies with radioactive leucine, isoleucine, valine and acetate showed that in Flavobacterium spec. strain C1/2 leucine can function as the precursor of the terminally branched alkyl substituents in flexirubin-type pigments.Part 25 in the series Investigations on Metabolites of Microorganisms. For Part 24 see H. Achenbach, A. Böttger-Vetter, D. Hunkler, E. Fautz and H. Reichenbach, Tetrahedron, in the press     

Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl-cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO₂R′) reagents where R′ is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding β-S-(β-aminoethanethiol) and β-S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.     

Biodegradability and biodegradation pathways of endosulfan and endosulfan sulfate

Endosulfan and endosulfan sulfate are persistent organic pollutants that cause serious environmental problems. Although these compounds are already prohibited in many countries, residues can be detected in soils with a history of endosulfan application. Endosulfan is transformed in the environment into endosulfan sulfate, which is a toxic and persistent metabolite. However, some microorganisms can degrade endosulfan without producing endosulfan sulfate, and some can degrade endosulfan sulfate. Therefore, biodegradation has the potential to clean up soil contaminated with endosulfan. In this review, we provide an overview of aerobic endosulfan degradation by bacteria and fungi, and a summary of recent advances and prospects in this research field.     

Quantifying the biodegradation of phenanthrene by Pseudomonas stutzeri P16 in the presence of a nonionic surfactant.

The low water solubility of polycyclic aromatic hydrocarbons is believed to limit their availability to microorganisms, which is a potential problem for bioremediation of polycyclic aromatic hydrocarbon-contaminated sites. Surfactants have been suggested to enhance the bioavailability of hydrophobic compounds, but both negative and positive effects of surfactants on biodegradation have been reported in the literature. Earlier, we presented mechanistic models of the effects of surfactants on phenanthrene dissolution and on the biodegradation kinetics of phenanthrene solubilized in surfactant micelles. In this study, we combined the biodegradation and dissolution models to quantify the influence of the surfactant Tergitol NP-10 on biodegradation of solid-phase phenanthrene by Pseudomonas stutzeri P16. Although micellized phenanthrene does not appear to be available directly to the bacterium, the ability of the surfactant to increase the phenanthrene dissolution rate resulted in an overall increase in bacterial growth rate in the presence of the surfactant. Experimental observations could be predicted well by the derived model with measured biokinetic and dissolution parameters. The proposed model therefore can serve as a base case for understanding the physical-chemical effects of surfactants on nonaqueous hydrocarbon bioavailability.     

Anaerobic digestion of alcohol sulfate (anionic surfactant) rich wastewater--batch experiments. Part I: influence of the surfactant concentration

Textile wet processing wastewater (e.g., from cotton desizing) contains high concentrations of surfactants as well as readily biodegradable compounds like starch and other carbohydrates. Decyl sulfate (DS, surfactant) and soluble starch were used as model pollutants for biodegradation batch experiments. Very high loadings of the biomass were applied (DS: 21.7-217 g/kg cell dry weight (CDW); starch: 910 g/kg cell dry weight) to study inhibitory effects of the surfactant on the degradation cascade of the biopolymer. The starch hydrolysis was inhibited above sludge loadings of 65 g DS/kg CDW. Acidogenesis was the degradation step with the highest resistance towards inhibitory effects of the surfactant, whereas methanogenesis proved to be the most sensitive. The effects of the surfactant were described by the change of the methane evolution, which was reduced by 50% in 87 days with an addition of 58 g DS/kg CDW. The surfactant caused a high temporary accumulation of intermediates like volatile fatty acids. At the highest loading (217 g DS/kg CDW) the conversion of the substrate to methane was only minor.     

表面活性剂TW-80对土壤中多环芳烃生物降解的影响

以表面活性剂TW80为供试物,进行了为期150d的实验研究,并分别在30、60和150d间隔采样监测PAHs降解率。结果表明,30d后,土壤中PAHs的降解率达90%,比对照提高约30%.60d后,浓度为10000mg·kg^-1表面活性剂的土壤和对照中,PAHs降解率从65.1%和60%迅速提高到93.8%和79.2%.其它处理中,PAHs的平均降解率仅比30d的结果提高4%.150d后,所有处理中PAHs的降解率均达到90%以上。可以认为,表面活性剂能提高PAHs的生物可利用性,加快PAHs的降解速率,从而减少污染暴露时间。但表面活性剂浓度过高可抑制微生物活性。研究还发现,TW80土壤中含有优势真菌。经鉴定为常见青霉、蠕形青霉、淡紫青霉和顶孢头孢霉。它们是土壤PAHs迅速降解的动因.     

Primary biodegradation of a series of alkyl sulfosuccinates by mixed bacterial culture

A mixed bacterial culture capable of primary biodegradation of sodium alkyl sulfosuccinates R¹-OOC−CH(SO₃Na)−CH₂−COO−R² was obtained from soil microorganisms by enrichment cultivation and adaptation in the presence of mono-n-dodecyl sulfosuccinate. Gram-negative psychrophilic bacteria with proteolytic, lipolytic and ammonifying activities were prevalent in the culture. The process of primary biodegradation of alkyl sulfosuccinates can be described by firstorder reaction kinetics. The rate constants for linear esters were ascending in the order C₄<C₅<C₆ (45 μmol/min per g cell protein) and further descending with increasing length of the carbon chain C₆>C₈>=C₁₃. Substitution of cyclohexyl for n-hexyl group resulted in fourfold decrease in biodegradation rate. Terminal branching of alkyl chain does not affect the rate of primary biodegradation.     

Use of sodium dodecyl sulphate for stimulation of biodegradation of n-alkanes without residual contamination by the surfactant

The capacity of a range of aliphatic alkanes (C₆–C₁₆), intermediates of n-decane oxidation and sodium dodecyl sulphate (SDS) to induce decane-mineralization activity in the cells of Pseudomonas C12B was compared with that for n-decane. The comparison on quantitative basis had two serious limitations: low solubility of tested inducers in aqueous solutions and their toxicity to bacterial cells. Carbon chain length and the presence of hydroxyl group were the important factors for induction activity. However, presence of hydroxyl groups at both ends of alkyl chain prevented the induction of decane-mineralization activity. Good induction activity by SDS was caused either by the presence of free end of alkyl chain, or by bacterial hydrolysis of sulphate group to yield alcohol, which in turn served as true inducer. The presence of SDS in the culture medium with n-decane as main source of carbon and energy accelerated the growth of Pseudomonas C12B. SDS disappeared from the culture medium in early stages of cultivation suggesting preferential degradation by the bacterium, while the consumption of n-decane was accelerated. This may be associated with the capacity of SDS to induce decane-mineralization system in Pseudomonas C12B and/or with the ability of SDS to stimulate the surface attachment of competent bacteria resulting in the close proximity of the cells with alkane droplets, and thus, enhanced breakdown of the hydrocarbon pollutant.