Characterization of fluoranthene- and pyrene-degrading bacteria isolated from PAH-contaminated soils and sediments

Sixteen environmental samples, from the United States, Germany and Norway, with histories of previous exposure to either creosote, diesel fuel or coal tar materials, were screened for bacteria which could degrade high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A modified version of the spray plate technique was used for the isolations. Using fluoranthene (FLA) and pyrene (PYR) as model HMW PAHs, we isolated 28 strains on FLA and 21 strains on PYR. FLA degraders were defined as able to grow on FLA but not PYR. PYR degraders grew on both PAHs. All PYR degraders were found to be Gram-positive and all FLA degraders were Gram-negative. GC-FAME analysis showed that many of the PYR degraders were Mycobacterium spp and many of the FLA degraders were Sphingomonas spp. Comparison of the metabolic characteristics of the strains using the spray plate technique and direct growth studies revealed that more than half of the FLA degraders (59%) were able to cometabolize PYR (ie, they produced clearing zones or colored metabolites on spray plates but did not grow on the PAH) and the ability of many of these strains to cometabolize fluorene, anthracene, benzo[b]fluorene, benzo[a]anthracene and benzo[a]pyrene was significantly affected by pre-exposure to phenanthrene. Studies on the metabolic products produced from PYR cometabolism by strain EPA 505 suggested the possibility of attack at two different sites on the PYR molecule. However, the inability to derive degradable carbon from initial opening of one of the PYR rings probably accounted for the lack of growth on this PAH by the FLA-degrading strains. The PYR degraders on the other hand, were less able to cometabolize HMW PAHs, even following pre-exposure to PHE. Characterization of the FLA degradation pathway for several of the Sphingomonas isolates indicated oxidation and ring opening through to acenaphthenone as the principle metabolite. Strain CO6, however, also oxidized FLA through fluorenone, suggesting a dual attack on the FLA molecule, similar to that observed by others in Mycobacterium spp. Journal of Industrial Microbiology & Biotechnology (2000) 24, 100–112. Received 01 May 1999/ Accepted in revised form 01 November 1999     

Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and phenanthrene by Sphingomonas paucimobilis var. EPA505

Catabolic pathways for utilization of naphthalene (NAP), anthracene (ANT), phenanthrene (PHE), and fluoranthene (FLA) by Sphingomonas paucimobilis EPA505 were identified. Accumulation of catabolic intermediates was investigated with three classes of Tn5 mutants with the following polycyclic aromatic hydrocarbon (PAH)-negative phenotypes; (class I NAP(-) PHE(-) FLA(-), class II NAP(-) PHE(-), and class III FLA(-)). Class I mutant 200pbhA had a Tn5 insertion within a meta ring fission dioxygenase (pbhA), and a ferredoxin subunit gene (pbhB) resided directly downstream. Mutant 200pbhA and other class I mutants lost the ability to catalyze the initial dihydroxylation step and did not transform NAP, ANT, PHE, or FLA. Class I mutant 401 accumulated salicylic acid, 2-hydroxy-3-naphthoic acid, 1-hydroxy-2-naphthoic acid, and hydroxyacenaphthoic acid during incubation with NAP, ANT, PHE, or FLA, respectively. Class II mutant 132pbhC contained the Tn5 insertion in an aldolase hydratase (pbhC) and accumulated what appeared to be meta ring fission products: trans-o-hydroxybenzylidene pyruvate, trans-o-hydroxynaphylidene pyruvate, and trans-o-hydroxynaphthyl-oxobutenoic acid when incubated with NAP, ANT, and PHE, respectively. When mutant 132pbhC was incubated with 1-hydroxy-2-naphthoic acid, it accumulated trans-o-hydroxybenzylidene pyruvate. Class III mutant 104ppdk had a Tn5 insertion in a pyruvate phosphate dikinase gene that affected expression of a FLA-specific gene and accumulated a proposed meta ring fission product; trans-o-hydroxyacenaphyl-oxobutenoic acid during incubation with FLA. Trans-o-hydroxyacenaphyl-oxobutenoic acid was degraded to acenaphthenone that accumulated with class III mutant 611. Acenaphthenone was oxidized via incorporation of one molecule of dioxygen by another oxygenase. 2,3-Dihydroxybenzoic acid was the final FLA-derived catabolic intermediate detected. Analysis of PAH utilization mutants revealed that there are convergent and divergent points involved in NAP, ANT, PHE, and FLA utilization by S. paucimobilis EPA505.     

Biodegradation kinetics of select polycyclic aromatic hydrocarbon (PAH) mixtures by <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> EPA505

Many contaminated sites commonly have complex mixtures of polycyclic aromatic hydrocarbons (PAHs) whose individual microbial biodegradation may be altered in mixtures. Biodegradation kinetics for fluorene, naphthalene, 1,5-dimethylnaphthalene and 1-methylfluorene were evaluated in sole substrate, binary and ternary systems using Sphingomonas paucimobilis EPA505. The first order rate constants for fluorene, naphthalene, 1,5-dimethylnaphthalene, and 1-methylfluorene were comparable; yet Monod parameters were significantly different for the tested PAHs. S. paucimobilis completely degraded all the components in binary and ternary mixtures; however, the initial degradation rates of individual components decreased in the presence of competitive PAHs. Results from the mixture experiments indicate competitive interactions, demonstrated mathematically. The generated model appropriately predicted the biodegradation kinetics in mixtures using parameter estimates from the sole substrate experiments, validating the hypothesis of a common rate-determining step. Biodegradation kinetics in mixtures were affected by the affinity coefficients of the co-occurring PAHs and mixture composition. Experiments with equal concentrations of substrates demonstrated the effect of concentration on competitive inhibition. Ternary experiments with naphthalene, 1,5-dimethylnaphthalene and 1-methylfluorene revealed delayed degradation, where depletion of naphthalene and 1,5-dimethylnapthalene occurred rapidly only after the complete removal of 1-methylfluorene. The substrate interactions observed in mixtures require a multisubstrate model to account for simultaneous degradation of substrates. PAH contaminated sites are far more complex than even ternary mixtures; however these studies clearly demonstrate the effect that interactions can have on individual chemical kinetics. Consequently, predicting natural or enhanced degradation of PAHs cannot be based on single compound kinetics as this assumption would likely overestimate the rate of disappearance.     

Degradation of selected (bio-)surfactants by bacterial cultures monitored by calorimetric methods

The subjects of the article are investigations concerning the ability of both Rhodococcus opacus 1CP and mixed bacterial cultures to use selected surfactants as sole carbon and energy source. In a comparative manner the biosurfactants rhamnolipid, sophorolipid and trehalose tetraester, and the synthetic surfactant Tween 80 were examined. Particular emphasis was put on a combinatorial approach to determine quantitatively the degree of surfactant degradation by applying calorimetry, thermodynamic calculations and mass spectrometry, HPLC as well as determination of biomass. The pure bacterial strain R. opacus was only able to metabolize a part of the synthetic surfactant Tween 80, whereas the mixed bacterial cultures degraded all of the applied surfactants. Exclusive for the biosurfactant rhamnolipid a complete microbial degradation could be demonstrated. In the case of the other surfactants only primary degradation was observed.     

Measurement of biodegradability parameters for single unsubstituted and methylated polycyclic aromatic hydrocarbons in liquid bacterial suspensions

Substrate depletion experiments were conducted to characterize aerobic biodegradation of 20 single polycyclic aromatic hydrocarbons (PAHs) by induced Sphingomonas paucimobilis strain EPA505 in liquid suspensions. PAHs consisted of low molecular weight, unsubstituted, and methyl-substituted homologs. A material balance equation containing the Andrews kinetic model, an extension of the Monod model accounting for substrate inhibition, was numerically fitted to batch depletion data to estimate extant kinetic parameters including the maximal specific uptake rates, q(max), the affinity coefficients, K(S), and the substrate inhibition coefficients, K(I). Strain EPA505 degraded all PAHs tested. Applied kinetic models adequately simulated experimental data. A cell proliferation assay involving reduction of the tetrazolium dye WST-1 was used to evaluate the ability of strain EPA505 to utilize individual PAHs as sole energy and carbon sources. Of the 22 PAHs tested, 9 supported bacterial growth. Evaluation of the biokinetic data showed that q(max) correlated highly with transmembrane flux as theoretically estimated by a diffusion model, pointing to transmembrane transport as a potential rate-determining process. The biodegradability data generated in this study is essential for the development of quantitative structure-activity relationships (QSARs) for biodegradability and for modeling biodegradation of simple PAH mixtures.     

表面活性剂对分枝杆菌KR2菌株降解菲的影响

采用同位素示踪方法,从表面活性剂的浓度、离子类型和直链长度三方面研究了表面活性剂对分枝杆菌KR2菌株降解菲的影响。结果表明,表面活性剂的存在不能促进KR2菌对菲的降解;高浓度表面活性剂(≥20mg·L^-1)的存在,使菲的降解出现延迟期,非离子表面活性剂Tween80在低浓度时(≤10mg·L^-1)可以优先作为营养基质被分枝杆菌KR2菌株利用,表面活性剂的离子类型对菲降解的抑制作用的顺序为阳离子表面活性剂TDTMA>阴离子表面活性剂LAS>非离子表面活性剂Tween80,表面活性剂的直链长度对菲降解的影响为直链越短,对微生物的毒性越大,菲降解得越不完全。     

Biodegradation of phenanthrene by Pseudomonas sp. BZ-3, isolated from crude oil contaminated soil

A phenanthrene (PHE) degrading bacterium strain BZ-3 was isolated from the crude oil contaminated soil in Binzhou, China. The isolate was identified as Pseudomonas sp. BZ-3 on the basis of 16S rRNA gene sequence. Various experiments were conducted to investigate the effect of pH, salinity and PHE concentration on the degradation efficiency of PHE. The degradation efficiency and degradation metabolites of PHE were detected by using GC–MS and HPLC-MS analyses. The strain BZ-3 could degrade 75% of PHE at an initial concentration of 50 mg/L under 20 g/L salinity in 7 days. PHE degradation kinetics was estimated in a first-order degradation rate model and the rate coefficient was calculated as 0.108 d⁻¹. On the basis of the identified degradation metabolites, the strain BZ-3 could degrade PHE in the salicylate metabolic pathway. In a mixture system consisting of PHE and other PAHs including naphthalene (NA), anthracene (ANTH), and pyrene (PYR), the strain BZ-3 showed an efficiently degradation capability. Further study showed that the strain BZ-3 could also use NA, ANTH, PYR, xylene, 1-hydroxy-2-naphthoic acid, and hexane as the sole carbon and energy source, but did not grow on nitrobenzene-containing medium.     

Determination of polycyclic aromatic hydrocarbons in urine of coke oven workers by headspace solid phase microextraction and gas chromatography-mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) represent a complex mixture of toxic compounds that are ubiquitous in the environment. We investigated the utility of head space-solid phase microextraction (HS-SPME) to measure the following surrogate PAHs in urine: naphthalene (NAP), phenanthrene (PHE), pyrene (PYR), and benzo(a)pyrene (BAP), representing classes of 2-, 3-, 4- and 5-ring compounds, respectively. We then applied the method to urine from 28 coke oven workers (median levels (microg/l) were: NAP=3.65, PHE=1.51, PYR=0.003, BAP not detected) and 22 controls (median (microg/l) NAP=0.859, PHE=0.062, PYR=0.001, BAP not detected). Urinary levels of NAP, PHE, and PYR were all associated with exposure category (controls, side- and bottom-workers, and top-workers) but not with smoking status. Strong correlations were observed between urinary levels of NAP, PHE, and PYR in coke-oven workers. Our results indicate that unmetabolized 2-, 3- and 4-ring PAHs can be measured in urine by HS-SPME. Such measurements can be used to investigate the uptake and metabolism of complex PAH mixtures in humans.     

Degradation of 2,4,6-trinitrotoluene by Serratia marcescens

A strain of Serratia marcescens, isolated from the soil of a contaminated site, degraded 2,4,6-trinitrotoluene (TNT) as the sole source of carbon and energy. At an initial concentration of 50mg , TNT was totally degraded in 48h under aerobic conditions in a minimal salt medium. Reduction intermediates (4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene) were observed. The presence of a surfactant (Tween 80) is essential to facilitate rapid degradation.     

Direct Observation of the Photodegradation of Anthracene and Pyrene Adsorbed onto Mangrove Leaves

An established synchronous fluorimetry method was used for in situ investigation of the photodegradation of pyrene (PYR) and anthracene (ANT) adsorbed onto fresh leaves of the seedlings of two mangrove species, Aegiceras corniculatum (L.) Blanco (Ac) and Kandelia obovata (Ko) in multicomponent mixtures (mixture of the ANT and PYR). Experimental results indicated that photodegradation was the main transformation pathway for both ANT and PYR in multicomponent mixtures. The amount of the PAHs volatilizing from the leaf surfaces and entering the inner leaf tissues was negligible. Over a certain period of irradiation time, the photodegradation of both PYR and ANT adsorbed onto the leaves of Ac and Ko followed first-order kinetics, with faster rates being observed on Ac leaves. In addition, the photodegradation rate of PYR on the leaves of the mangrove species in multicomponent mixtures was much slower than that of adsorbed ANT. Compared with the PAHs adsorbed as single component, the photodegradation rate of ANT adsorbed in multicomponent mixtures was slower, while that of PYR was faster. Moreover, the photodegradation of PYR and ANT dissolved in water in multicomponent mixtures was investigated for comparison. The photodegradation rate on leaves was much slower than in water. Therefore, the physical-chemical properties of the substrate may strongly influence the photodegradation rate of adsorbed PAHs.     

Degradation of a mixture of high‐molecular‐weight polycyclic aromatic hydrocarbons by a Mycobacterium strain PYR‐1

Mycobacterium sp. PYR‐1, which was previously shown to mineralize several individual polycyclic aromatic hydrocarbons (PAHs), simultaneously degraded phenanthrene, anthracene, fluoranthene, pyrene and benzo[a]pyrene in a six‐component synthetic mixture. Chrysene was not degraded significantly. When provided with a complex carbon source, Mycobacterium sp. PYR‐1 degraded greater than 74% of the total PAH mixture during 6 d of incubation. Mycobacterium sp. PYR‐1 appeared to preferentially degrade phenanthrene. No significant difference in degradation rates was observed between fluoranthene and pyrene. Anthracene degradation was slightly delayed but, once initiated, proceeded at a constant rate. Benzo[a]pyrene was degraded slowly. Degradation of a crude mixture of benzene‐soluble PAHs from contaminated sediments resulted in a 47% reduction of the material in 6 d compared with that of autoclaved controls. Experiments using an environmental microcosm test system indicated that mineralization rates of individual ¹⁴C‐labeled compounds were significantly lower in the mixtures than in equivalent doses of these compounds alone. Mineralization of the complete mixture was estimated conservatively to be between 49.7 and 53.6% and was nearly 50% in 30 d of incubation when all compounds were radiolabeled. These results strengthen the argument for the potential application of Mycobacterium sp. PYR‐1 for bioremediation of PAH‐contaminated wastes.     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.     

Enhanced esterification activity through interfacial activation and cross‐linked immobilization mechanism of Rhizopus oryzae lipase in a nonaqueous medium

Interfacial activation via surfactant (Tween 80, Triton X‐100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross‐linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non‐pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899–904, 2016     

共基质对10株细菌降解芘的作用

从石油污染的污泥中分离出10株细菌(SB01—SB10),研究了有(或无)共基质(葡萄糖Glu,或菲PHE)对细菌降解芘(PYR)的影响.结果表明：当以PYR为唯一碳源和能源时(MS₁),SB01的PYR降解率最高,5 d可降解30.4%;以Glu为共代谢基质时(MS₂),SB09的PYR降解率最高,可达37.7%;以PHE为共代谢基质时(MS₃),SB10的PYR降解率为50.2%.Glu抑制SB01、SB03对PYR的降解,对SB01抑制作用最明显,使SB01的PYR降解率降低7.9%;Glu对SB02、SB07、SB08、SB10降解率无明显促进或抑制作用.PHE对细菌降解PYR均有促进作用,对SB10的促进作用最明显,使其降解率提高298%.Glu与PHE对SB04和SB09降解PYR的促进作用无显著差异,而对其它各菌株而言,PHE对PYR降解的促进作用大于Glu.     

Effects of monorhamnolipid and Tween 80 on the degradation of phenol by Candida tropicalis

The effects of the biosurfactant monorhamnolipid (monoRL) and the chemical surfactant Tween 80 on the degradation of phenol by Candida tropicalis CICC 1463 were studied. Both surfactants impeded the decay in cell concentrations at the beginning of the fermentation and enhanced the cell growth thereafter. They also increased the degradation efficiencies of 500 mg/L phenol from 86.9% in control to above 99.0% for all test concentrations within 30 h. The monoRL could also be degraded by the C. tropicalis. These results indicate that the surfactants could diminish the toxicity of phenol to the yeast, increase cell growth and improve phenol removal. The monoRL is better than Tween 80 because of biodegradability.     

Predicting stability of mixed microbial cultures from single species experiments: 1. Phenomenological model

The growth of mixed microbial cultures on mixtures of substrates is a fundamental problem of both theoretical and practical interest. On the one hand, the literature is abundant with experimental studies of mixed-substrate phenomena [T. Egli, The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates, Adv. Microbiol. Ecol. 14 (1995) 305-386]. On the other hand, a number of mathematical models of mixed-substrate growth have been analyzed in the last three decades. These models typically assume specific kinetic expressions for substrate uptake and biomass growth rates and their predictions are formulated in terms of parameters of the model. In this work, we formulate and analyze a general mathematical model of mixed microbial growth on mixtures of substitutable substrates. Using this model, we study the effect of mutual inhibition of substrate uptake rates on the stability of the equilibria of the model. Specifically, we address the following question: How much of the dynamics exhibited by two competing species can be inferred from single species data? We provide geometric criteria for stability of various types of equilibria corresponding to non-competitive exclusion, competitive exclusion, and coexistence of two competing species in terms of growth isoclines and consumption curves. A growth isocline is a curve in the plane of substrate concentrations corresponding to the zero net growth of a given species. In [G.T. Reeves, A. Narang, S.S. Pilyugin, Growth of mixed cultures on mixtures of substitutable substrates: The operating diagram for a structured model, J. Theor. Biol. 226 (2004) 143-157], we introduced consumption curves as sets of all possible combinations of substrate concentrations corresponding to balanced growth of a single microbial species. Both types of curves can be obtained in single species experiments.     

The stimulatory effects of surfactants on composting of waste rich in cellulose

The chemical surfactant Tween 80 and biosurfactant rhamnolipid were respectively added to the composting substrate, a mixture of rice straw and bran, and their effects on the composting process were investigated. Samples were analysed for microbial communities of bacteria, actinomycetes and fungi, carboxymethylcellulose hydrolysis (CMCase) and xylanase activities, cellulose and hemicellulose fractions, water-soluble carbon (WSC) contents in the substrates, organic matter contents and pH values during the composting process. The results showed that both Tween 80 and rhamnolipid had slight stimulatory effects on the microbial populations of bacteria, actinomycetes and fungi. In addition, rhamnolipid increased the peak xylanase activity 15% higher than that of the control, while Tween 80 increased the maximum CMCase activity 35% higher than that of the control. As a result of the increased enzyme activities, treatments with Tween 80 and rhamnolipid were of higher WSC contents than the control during the whole composting process. Accordingly, the composting process was accelerated by the surfactants, since the organic matter was decomposed more quickly and the breakdown of cellulose and hemicellulose was better in the treatments with Tween 80 and rhamnolipid.     

Utilization of solubilized and crystalline mixtures of polycyclic aromatic hydrocarbons by a Mycobacterium sp.

A strain of Mycobacterium, that is able to degrade fluorene, phenanthrene, fluoranthene and pyrene was grown on various mixtures of these substrates. The polycyclic aromatic hydrocarbons (PAH) were provided either as crystals or solubilized by a surfactant. Mixed PAH were degraded simultaneously, but not in parallel, indicating that the degradation pathways were not incompatible. Certain interactions of the substrates were observed. For example, the degradation of solubilized pyrene was delayed in the presence of fluorene and enhanced in the presence of phenanthrene. Fluorene was degraded cometabolically with the other PAH serving as growth substrates, but not as the only source of carbon. The utilization of phenanthrene occurred at the fastest rate and was not affected by the presence of fluorene, pyrene or fluoranthene.     

Impact of Clay Minerals and DOM on the Competitive Sorption/Desorption of PAHs

Two model compounds were used to investigate sorptive phenomena of a silty-sand soil under single and binary solute systems at different concentrations. In the sorption isotherms, the presence of phenan-threne (PHE) exhibited a statistically significant (P<0.05) sorption competition over pyrene (PYR), regardless of the concentration. PYR influenced the PHE isotherms only when it was present at 15?mg/L. The concentration dependence in sorption competition was only evident for the more hy-drophobic PYR. In the presence of PHE, the fraction of desorbed PYR was significantly increased with an increase in PYR concentration. PHE desorption enhancement was the most observable with the higher initial concentration (15?mg/L). However, the presence of PYR did not affect PHE desorption. This study found that, based on equivalent solid mass, soils containing only clay minerals sorbed 12.2% more PHE than soils with only soil organic matter (SOM) for 3?mg/L PHE. Clay minerals also impacted desorption as evidenced by a 65% decrease in desorbed PYR fraction compared with when the soil only contained SOM. The dissolved organic matter (DOM) amendment did not increase desorption. Instead, PHE desorption was significantly inhibited by the added DOM. For this study, co-sorption was found to be the mechanism for the inhibited desorption.