Influence of the growth substrate on ester-linked phospho- and glycolipid fatty acids of PAH-degrading Mycobacterium sp. LB501T

The influences of poorly water-soluble anthracene on ester-linked phospholipid fatty acid (PLFA) and glycolipid fatty acid (GLFA) profiles of Mycobacterium sp. LB501T were studied. Bacteria were cultivated on either anthracene or glucose (one culture with successively amended small doses of this substrate and one with excess concentrations) to distinguish between influences of the chemical structure and the bioavailability of the growth substrate. Results revealed that GLFA and PLFA profiles of M. sp. LB501T depended on the availability and the structure of the carbon source. Fatty acid profiles obtained with anthracene differed from those obtained with excess glucose. They were interpreted as a specific adaptation to this poorly bioavailable polycyclic aromatic hydrocarbon (PAH). In contrast, profiles obtained with low glucose concentrations showed clear signs of starvation stress. Stable carbon isotopic ratios (delta13C) of GLFA and PLFA of M. sp. LB501T were analysed to characterize the 13C-fractionation during the biosynthesis of individual fatty acids and to evaluate their value as markers for substrate usage. Although the delta13C values of PLFA and GLFA showed differential isotope fractionation during anthracene- and glucose-degradation, they were sufficiently distinct to be used as signatures of bacterial substrate usage.     

Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway,through o-phthalic acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Characterization of multiple-substrate utilization by anthracene-degrading Mycobacterium frederiksbergense LB501T

Stable carbon isotope analysis of biomass and analyses of phospholipid fatty acids (PLFA), glycolipid fatty acids (GLFA), and mycolic acids were used to characterize mixed-substrate utilization by Mycobacterium frederiksbergense LB501T under various substrate regimens. The distinct (13)C contents of anthracene and glucose as representatives of typical hydrophobic pollutants and naturally occurring organic compounds, respectively, were monitored during formation into biomass and used to quantify the relative contributions of the two carbon sources to biomass formation. Moreover, the influence of mixed-substrate utilization on PLFA, GLFA, and mycolic acid profiles and cell surface hydrophobicity was investigated. Results revealed that M. frederiksbergense LB501T degrades anthracene and forms biomass from it even in the presence of more readily available dissolved glucose. The relative ratios of straight-chain saturated PLFA to the corresponding unsaturated PLFA and the total fraction of saturated cyclopropyl-branched PLFA of M. frederiksbergense LB501T depended on the carbon source and the various rates of addition of mixed substrates, whereas no such trend was observed with GLFA. Higher proportions of anthracene in the carbon source mixture led to higher cell surface hydrophobicities and more-hydrophobic mycolic acids, which in turn appeared to be valuable indicators for substrate utilization by M. frederiksbergense LB501T. The capability of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria to utilize readily available substrates besides the poorly available PAHs favors the buildup of PAH-degrading biomass. Feeding of supplementary carbon substrates may therefore promote bioremediation, provided that it sustains the pollutant-degrading population rather than other members of the microbial community.     

Influence of the growth substrate on the mycolic acid profiles of mycobacteria

The influence of growth substrates on mycolic acid profiles of PAH-degrading Mycobacterium spp. LB501T, LB307T and VM552 was examined by high-performance liquid chromatography (HPLC) using glucose, alkanes, polycyclic aromatic hydrocarbons (PAH) or Luria-Bertani medium (LB) as the sole carbon source. The substrates gave rise to varying mycolic acid profiles, as bacteria growing on poorly water-soluble substrates exhibited more hydrophobic mycolic acids than cells grown on glucose. Our results indicate that mycobacteria respond to the growth substrate by changing the mycolic acid composition of their cell wall, pointing at the importance of the growth substrate for mycolic acid profiling as an identification method of actinomycetes.     

Characterization of Multiple-Substrate Utilization by Anthracene-Degrading Mycobacterium frederiksbergense LB501T

Stable carbon isotope analysis of biomass and analyses of phospholipid fatty acids (PLFA), glycolipid fatty acids (GLFA), and mycolic acids were used to characterize mixed-substrate utilization by Mycobacterium frederiksbergense LB501T under various substrate regimens. The distinct ¹³C contents of anthracene and glucose as representatives of typical hydrophobic pollutants and naturally occurring organic compounds, respectively, were monitored during formation into biomass and used to quantify the relative contributions of the two carbon sources to biomass formation. Moreover, the influence of mixed-substrate utilization on PLFA, GLFA, and mycolic acid profiles and cell surface hydrophobicity was investigated. Results revealed that M. frederiksbergense LB501T degrades anthracene and forms biomass from it even in the presence of more readily available dissolved glucose. The relative ratios of straight-chain saturated PLFA to the corresponding unsaturated PLFA and the total fraction of saturated cyclopropyl-branched PLFA of M. frederiksbergense LB501T depended on the carbon source and the various rates of addition of mixed substrates, whereas no such trend was observed with GLFA. Higher proportions of anthracene in the carbon source mixture led to higher cell surface hydrophobicities and more-hydrophobic mycolic acids, which in turn appeared to be valuable indicators for substrate utilization by M. frederiksbergense LB501T. The capability of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria to utilize readily available substrates besides the poorly available PAHs favors the buildup of PAH-degrading biomass. Feeding of supplementary carbon substrates may therefore promote bioremediation, provided that it sustains the pollutant-degrading population rather than other members of the microbial community.     

Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.     

Degradation of Anthracene by Mycobacterium sp. Strain LB501T Proceeds via a Novel Pathway, through o-Phthalic Acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Isolation of Adherent Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bacteria Using PAH-Sorbing Carriers

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.     

Surfactant inhibition of bacterial growth on solid anthracene

Surfactants have been proposed as a promising method to enhance bioremediation of hydrophobic compounds in contaminated soils. However, the results of effects of surfactants on bioremediation are not consistent. This study showed that Triton X-100 at low concentration (0.024 mM or 0.09 CMC) inhibited the rate of growth of either a Mycobacterium sp. or a Pseudomonas sp. on solid anthracene as sole carbon source. Recovery of microbial growth rate could be achieved by dilution of surfactants, while addition of more surfactant gave an immediate decrease in growth rate. No inhibition of growth by Triton X-100 was observed with growth on glucose. The surfactant sorbed onto the surfaces of both the cells and the anthracene particles, which could inhibit uptake of anthracene. The results were consistent with the hypothesis that inhibition of microbial adhesion of cells to anthracene was responsible for the inhibition of growth by Triton X-100.     

Use of the pAL5000 replicon in PAH-degrading mycobacteria: application for strain labelling and promoter probing

Three environmental Mycobacterium strains (LB501T, LB307T and VM552) able to degrade anthracene, phenanthrene or pyrene, respectively, were successfully electroporated with pAL5000-based plasmids containing the green fluorescent protein (gfp) gene of Aequoria victoria under the control of the hsp60 promoter of Mycobacterium bovis following a slightly modified standard procedure. Transformants showed irregular gfp expression profiles. Four plasmid derivatives were constructed that contained gene promoters isolated from, and adapted to, gene expression in polycyclic aromatic hydrocarbon (PAH)-degrading mycobacteria. One derivative directed strong and homogeneous expression of GFP, allowing dual analysis of both GFP- and PAH-derived fluorescence as assessed by confocal laser scanning microscopy. The results reported here demonstrate the suitability of the pAL5000 replicon for the development of recombinant DNA-based studies in PAH-degrading Mycobacterium spp.     

Fluorene and phenanthrene uptake by Pseudomonas putida ATCC 17514: kinetics and physiological aspects

Pseudomonas putida ATCC 17514 was used as a model strain to investigate the characteristics of bacterial growth in the presence of solid fluorene and phenanthrene. Despite the lower water-solubility of phenanthrene, P. putida degraded this polycyclic aromatic hydrocarbon (PAH) at a maximum observed rate of 1.4 +/- 0.1 mg L(-1) h(-1), higher than the apparent degradation rate of fluorene, 0.8 +/- 0.07 mg L(-1) h(-1). The role of physiological processes on the biodegradation of these PAHs was analyzed and two different uptake strategies were identified. Zeta potential measurements revealed that phenanthrene-grown cells were slightly more negatively charged (-57.5 +/- 4.7 mV) than fluorene-grown cells (-51.6 +/- 4.9 mV), but much more negatively charged than glucose-grown cells (-26.8 +/- 3.3 mV), suggesting that the PAH substrate induced modifications on the physical properties of bacterial surfaces. Furthermore, protein-to-exopolysaccharide ratios detected during bacterial growth on phenanthrene were typical of biofilms developed under physicochemical stress conditions, caused by the presence of sparingly water-soluble chemicals as the sole carbon and energy source for growth, the maximum value for TP/EPS during growth on phenanthrene (1.9) being lower than the one obtained with fluorene (5.5). Finally, confocal laser microscopy observations using a gfp-labeled derivative strain revealed that, in the presence of phenanthrene, P. putida::gfp cells formed a biofilm on accessible crystal surfaces, whereas in the presence of fluorene the strain grew randomly between the crystal clusters. The results showed that P. putida was able to overcome the lower aqueous solubility of phenanthrene by adhering to the solid PAH throughout the production of extracellular polymeric substances, thus promoting the availability and uptake of such a hydrophobic compound.     

Cross-induction of pyrene and phenanthrene in a Mycobacterium sp. isolated from polycyclic aromatic hydrocarbon contaminated river sediments.

A polycyclic aromatic hydrocarbon (PAH)-degrading culture enriched from contaminated river sediments and a Mycobacterium sp. isolated from the enrichment were tested to investigate the possible synergistic and antagonistic interactions affecting the degradation of pyrene in the presence of low molecular weight PAHs. The Mycobacterium sp. was able to mineralize 63% of the added pyrene when it was present as a sole source of carbon and energy. When the enrichment culture and the isolated bacterium were exposed to phenanthrene, de novo protein synthesis was not required for the rapid mineralization of pyrene, which reached 52% in chloramphenicol-treated cultures and 44% in the absence of the protein inhibitor. In the presence of chloramphenicol, < 1% of the added pyrene was mineralized by the mixed culture after exposure to anthracene and naphthalene. These compounds did not inhibit pyrene utilization when present at the same time as pyrene. Concurrent mineralization of pyrene and phenanthrene after exposure to either compound was observed. Cross-acclimation between ring classes of PAHs may be a potentially important interaction influencing the biodegradation of aromatic compounds in contaminated environments.     

Limits of propidium iodide as a cell viability indicator for environmental bacteria.

BACKGROUND: Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. METHODS: Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. RESULTS: PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. CONCLUSIONS: The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.     

Enhanced biodegradation of phenanthrene by a marine bacterium in presence of a synthetic surfactant

The biodegradation of phenanthrene by the marine strain Sphingomonas sp. 2MPII (DSMZ 11572) was enhanced by the solubilizating properties of the nonionic surfactant Tween 80. After 197 h of incubation, 85 +/- 4% of the initial amount of phenanthrene (0.4 g l-1) was biodegraded in presence of Tween 80 (0.5 g l-1) as opposed to 52 +/- 5% without this synthetic surfactant. These results confirm that the activity of the strain 2MPII is limited by the bioavailability of the polycyclic aromatic hydrocarbon (PAH) substrate in the aqueous phase. Tween 80 appears to be efficient in increasing the bioavailability of hydrophobic compounds such as PAHs.     

Anthracene biodegradation and surface activity by an iron-stimulated Pseudomonas sp

Iron may enhance polycyclic aromatic hydrocarbons (PAHs) degradation directly by increasing the activity of the enzymes involved in the aerobic biodegradation pathways for hydrocarbons, and indirectly by increasing the PAHs bioavailability due to the stimulation of biosurfactant production. In the present work, the PAH anthracene was used in order to study the effect of different forms and concentrations of iron on its biodegradation and surfactant production by Pseudomonas spp. isolates from a 14-years old petrochemical sludge landfarm site. Among the iron forms, iron nitrate was chosen based on its high solubility and effect on the increase in the growth of the isolate. Iron concentration of 0.1mM was selected as the limit between deficiency and toxicity for isolates growth and anthracene degradation. After 48 days Pseudomonas citronellolis isolate 222A degraded 72% of anthracene related to iron stimulation and surface tension decrease, indicating surfactant production. Pseudomonas aeruginosa isolate 332C was iron-stimulated but did not reduce surface tension while P. aeruginosa isolate 312A exhibited a noniron and surfactant dependence to degrade 72% of anthracene. Isolate 222A showed a direct dependence on iron to stimulate surfactant activity, which probably increased anthracene bioavailability. To our knowledge, this is the first report about the iron effect on anthracene degradation and surfactant production by a Pseudomonas sp. Based on the iron requirement and surfactant activity, the Pseudomonas isolates may be useful for bioremediation of PAHs.     

Microbial consortium bioaugmentation of a polycyclic aromatic hydrocarbons contaminated soil

In this study we evaluated the capacity of a defined microbial consortium (five bacteria: Mycobacterium fortuitum, Bacillus cereus, Microbacterium sp., Gordonia polyisoprenivorans, Microbacteriaceae bacterium, Naphthalene-utilizing bacterium; and a fungus identified as Fusarium oxysporum) isolated from a PAHs contaminated landfarm site to degrade and mineralize different concentrations (0, 250, 500 and 1000 mg kg(-1)) of anthracene, phenanthrene and pyrene in soil. PAHs degradation and mineralization was evaluated by gas chromatography and respirometry, respectively. The microbial consortium degraded on average, 99%, 99% and 96% of the different concentrations of anthracene, phenanthrene and pyrene in the soil, in 70 days, respectively. This consortium mineralized 78%, on average, of the different concentrations of the 3 PAHs in soil after 70 days. Contrarily, the autochthonous soil microbial population showed no substantial mineralization of the PAHs. Bacterial and fungal isolates from the consortium, when inoculated separately to the soil, were less effective in anthracene mineralization compared to the consortium. This signifies synergistic promotion of PAHs mineralization by mixtures of the monoculture isolates (the microbial consortium).     

Enterobacter sp. VKGH12 growing with n-butanol as the sole carbon source and cells to which the alcohol is added as pure toxin show considerable differences in their adaptive responses

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is able to grow in toxic concentrations of n-butanol up to 1.5 % (volume in volume) as the sole carbon and energy source. Morphology changes in the cells growing on increasing concentrations of n-butanol were observed. The size of the bacteria decreased with increasing concentrations of n-butanol, also leading to an enhanced ratio between the surface and volume of the cells. This is in complete contradiction to the reaction of glucose-grown cells to which n-butanol had been added as a toxin. Similar differences were found in typical adaptive responses to toxic organic compounds, namely changes in fatty acid composition of membrane lipids and the activity of catalase. In both cases, reactions depending on the n-butanol concentrations could be observed when the toxin was added to glucose-grown cells, whereas no reaction was observable when the cells were growing in n-butanol as the sole carbon and energy source. This is another proof for the observation that there are certain differences between the adaptive strategies of cells when adapting to high concentrations of a growth substrate and those when adapting to a toxin added to growing cells.     

Adhesion and biofilm formation on polystyrene by drinking water-isolated bacteria

This study was performed in order to characterize the relationship between adhesion and biofilm formation abilities of drinking water-isolated bacteria (Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.). Adhesion was assessed by two distinct methods: thermodynamic prediction of adhesion potential by quantifying hydrophobicity and the free energy of adhesion; and by microtiter plate assays. Biofilms were developed in microtiter plates for 24, 48 and 72 h. Polystyrene (PS) was used as adhesion substratum. The tested bacteria had negative surface charge and were hydrophilic. PS had negative surface charge and was hydrophobic. The free energy of adhesion between the bacteria and PS was > 0 mJ/m² (thermodynamic unfavorable adhesion). The thermodynamic approach was inappropriate for modelling adhesion of the tested drinking water bacteria, underestimating adhesion to PS. Only three (B. cepacia, Sph. capsulata and Staphylococcus sp.) of the six bacteria were non-adherent to PS. A. calcoaceticus, Methylobacterium sp. and M. mucogenicum were weakly adherent. This adhesion ability was correlated with the biofilm formation ability when comparing with the results of 24 h aged biofilms. Methylobacterium sp. and M. mucogenicum formed large biofilm amounts, regardless the biofilm age. Given time, all the bacteria formed biofilms; even those non-adherents produced large amounts of matured (72 h aged) biofilms. The overall results indicate that initial adhesion did not predict the ability of the tested drinking water-isolated bacteria to form a mature biofilm, suggesting that other events such as phenotypic and genetic switching during biofilm development and the production of extracellular polymeric substances (EPS), may play a significant role on biofilm formation and differentiation. This understanding of the relationship between adhesion and biofilm formation is important for the development of control strategies efficient in the early stages of biofilm development.     

Bacterial degradation of benzalphthalide

APseudomonas sp., isolated by an enrichment culture technique, grew on benzalphthalide at up to 1 g/l as sole carbon source. Cells oxidized both benzalphthalide ando-phthalate at enhanced rates compared with glucose-grown cells, but catechol, gentisate and protocatechuate were oxidized slowly and equally by benzalphthalide-and glucose-grown cells.