An improved spectrophotometric method to study the transport, attachment, and breakthrough of bacteria through porous media

This study reports an improved spectrophotometric method for studying bacterial (Pseudomonas fluorescens UPER-1) transport and attachment in saturated porous media (silica sand). While studying the effect of ionic strength by the traditional packed-column spectrophotometric method, we encountered an artifact. The absorbance of a well-stirred bacterial suspension was found to decrease with time in the presence of high concentrations of sodium and potassium phosphate salts (> or = 10(-2) M) as the cells continued to age in a resting stage. Our results show that collision efficiency and a bed ripening index will be in error by as much as 20% if breakthrough is measured by the traditional spectrophotometric technique. We present an improved experimental technique that will minimize the artifact and should substantially advance the understanding of bacteria transport in porous media.     

Selection of Bacteria with Favorable Transport Properties Through Porous Rock for the Application of Microbial-Enhanced Oil Recovery

This paper presents a bench-scale study on the transport in highly permeable porous rock of three bacterial species—Bacillus subtilis, Pseudomonas putida, and Clostridium acetobutylicum—potentially applicable in microbial-enhanced oil recovery processes. The transport of cells during the injection of bacterial suspension and nutrient medium was simulated by a deep bed filtration model. Deep bed filtration coefficients and the maximum capacity of cells in porous rock were measured. Low to intermediate (~10⁶/ml) injection concentrations of cellular suspensions are recommended because plugging of inlet surface is less likely to occur. In addition to their resistance to adverse environments, spores of clostridia are strongly recommended for use in microbial-enhanced oil recovery processes since they are easiest among the species tested to push through porous rock. After injection, further transport of bacteria during incubation can occur by growth and mobility through the stagnant nutrient medium which fills the porous rock. We have developed an apparatus to study the migration of bacteria through a Berea sandstone core containing nutrient medium.     

Monitoring Bacterial Transport by Stable Isotope Enrichment of Cells

Understanding the transport and behavior of bacteria in the environment has broad implications in diverse areas, ranging from agriculture to groundwater quality, risk assessment, and bioremediation. The ability to reliably track and enumerate specific bacterial populations in the context of native communities and environments is key to developing this understanding. We report a novel bacterial tracking approach, based on altering the stable carbon isotope value (δ¹³C) of bacterial cells, which provides specific and sensitive detection and quantification of those cells in environmental samples. This approach was applied to the study of bacterial transport in saturated porous media. The transport of introduced organisms was indicated by mass spectrometric analysis of groundwater samples, where the presence of ¹³C-enriched bacteria resulted in increased δ¹³C values of the samples, allowing specific and sensitive detection and enumeration of the bacteria of interest. We demonstrate the ability to produce highly ¹³C-enriched bacteria, present data indicating that results obtained with this approach accurately represent intact introduced bacteria, and include field data on the use of this stable isotope approach to monitor in situ bacterial transport. This detection strategy allows sensitive detection of an introduced, unmodified bacterial strain in the presence of the indigenous bacterial community, including itself in its unenriched form.     

The use of a potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Uptake of l-malate and d-malate by luminal-membrane vesicles

The mechanisms of uptake of dicarboxylic acids by rabbit renal luminal-membrane vesicles were studied by the use of filtration and spectrophotometric techniques as described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J. 208, 359–368]. Addition of l- or d-malate to dye-membrane-vesicle suspensions in the presence of Na⁺ gradients (extravesicular>intravesicular) resulted in spectral curves indicative of depolarization events. The renal uptake of dicarboxylic acids was dependent on the type of Na⁺-salt anion present and could be correlated with the ability of the anions to penetrate biological membranes (i.e. Cl⁻>SO₄²⁻>gluconate). Identical results were obtained by a filtration technique with Sartorius membrane filters. The results indicate that the dicarboxylic acids are taken up by the membrane vesicles in an electrically positive form (i.e. Na⁺/substrate coupling ratio 3:1) by an Na⁺-dependent transport system. This proposal was further supported by spectrophotometric experiments with various ionophores such as valinomycin, gramicidin and nigericin. The absorbance changes associated with simultaneous addition of l- and d-malate and spectrophotometric competition studies revealed that the two isomers are taken up by a common transport system. Spectral changes of the dye induced by addition of increasing concentrations of l- or d-malate indicated that the transport system favours the unphysiological d-form rather than the l-form of malate. Furthermore, it was observed that the affinity of both isomers for the transport system was dependent on the concentration of Na⁺ in the medium.     

Purification and Properties of the F1Fo ATPase of Ilyobacter tartaricus, a Sodium Ion Pump

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F₁F_o ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the α, β, γ, , and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na⁺-translocating ATPase of Propionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and δ subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na⁺ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na⁺ or Li⁺ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na⁺, Li⁺, or H⁺. Proton transport was specifically inhibited by Na⁺ or Li⁺ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na⁺ as the physiological coupling ion for ATP synthesis.     

Polyphosphate Degradation in Stationary Phase Triggers Biofilm Formation via LuxS Quorum Sensing System in Escherichia coli

In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk⁻ppx⁻ or ppk⁻ppx⁻/ppk⁺ strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.     

Effect of Growth Conditions and Staining Procedure upon the Subsurface Transport and Attachment Behaviors of a Groundwater Protist

The transport and attachment behaviors of Spumella guttula (Kent), a nanoflagellate (protist) found in contaminated and uncontaminated aquifer sediments in Cape Cod, Mass., were assessed in flowthrough and static columns and in a field injection-and-recovery transport experiment involving an array of multilevel samplers. Transport of S. guttula harvested from low-nutrient (10 mg of dissolved organic carbon per liter), slightly acidic, granular (porous) growth media was compared to earlier observations involving nanoflagellates grown in a traditional high-nutrient liquid broth. In contrast to the highly retarded (retardation factor of ~3) subsurface transport previously reported for S. guttula, the peak concentration of porous-medium-grown S. guttula traveled concomitantly with that of a conservative (bromide) tracer. About one-third of the porous-medium-grown nanoflagellates added to the aquifer were transported at least 2.8 m downgradient, compared to only ~2% of the broth-grown nanoflagellates. Flowthrough column studies revealed that a vital (hydroethidine [HE]) staining procedure resulted in considerably less attachment (more transport) of S. guttula in aquifer sediments than did a staining-and-fixation procedure involving 4′,6′-diamidino-2-phenylindole (DAPI) and glutaraldehyde. The calculated collision efficiency (~10⁻² for porous-medium-grown, DAPI-stained nanoflagellates) was comparable to that observed earlier for the indigenous community of unattached groundwater bacteria that serve as prey. The attachment of HE-labeled S. guttula onto aquifer sediment grains was independent of pH (over the range from pH 3 to 9) suggesting a primary attachment mechanism that may be fundamentally different from that of their prey bacteria, which exhibit sharp decreases in fractional attachment with increasing pH. The high degree of mobility of S. guttula in the aquifer sediments has important ecological implications for the protistan community within the temporally changing plume of organic contaminants in the Cape Cod aquifer.     

Elevated Abundance of Bacteriophage Infecting Bacteria in Soil

Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 × 10⁷ g⁻¹), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 × 10⁸ g⁻¹, which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.     

Laboratory diagnosis of melioidosis: Past,present and future

Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized “in house” assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B. pseudomallei culture supernatants can be potentially distinguished from those of related bacterial species including B. thailandensis. Further studies using bacterial cultures and direct patient samples are required to evaluate the potential of metabolomics for improving diagnosis of melioidosis.     

The Suitability of a Quantitative Spectrophotometric Assay for Phenylalanine Ammonia-lyase Activity in Barley, Buckwheat, and Pea Seedlings

It has been suggested by others that the spectrophotometric assay of phenylalanine ammonia-lyase based on changes in absorbance at 290 nm may be complicated by a side reaction involving transamination from phenylalanine onto α-keto acids. This would lead to the production of phenylpyruvate which would spontaneously tautomerize and form an enol borate complex absorbing at this wavelength. We find that the inclusion of 1 ml of either 60 μm α-ketoglutarate or 500 μm phenylpyruvate in our 3-ml reaction mixtures has no significant effect on the spectrophotometric assay of phenylalanine ammonia-lyase in shoots from young seedlings of barley (Hordeum vulgare), buckwheat (Fagopyrum esculentum), or pea (Pisum sativum). Although these side reactions may be involved in preparations with very low enzyme activity, the spectrophotometric determination of phenylalanine ammonia-lyase based on changes in absorbance at 290 nm appears to be a reliable and sensitive technique in these seedlings.     

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca²⁺ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca²⁺ and Mn²⁺ ions. We show here that addition of Mn²⁺ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca²⁺. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca²⁺-deficient media is overcome by expression of other Ca²⁺ pumps, including a SERCA-type Ca²⁺ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca²⁺ and Mn²⁺ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.     

Effects of Glucose Concentrations on Cadmium, Copper, Mercury, and Zinc Toxicity to a Klebsiella sp

The influence of glucose concentration on Cd, Cu, Hg, and Zn toxicity to a Klebsiella sp. was studied by following the degradation of ¹⁴C-labeled glucose at pH 6.0. Uptake of ¹⁴C into the cells was also determined. The carbon concentrations ranged from 0.01 to 40 mg liter⁻¹, which are equivalent to soluble C concentrations in natural environments. The toxicity of Cu, Cd, and Zn to a Klebsiella sp. was affected considerably by the C concentration. Copper at 10⁻⁵ M was toxic when the carbon concentration was 10 or 40 mg liter⁻¹, while at 0.01 to 1.0 mg liter⁻¹ no toxicity was observed. Cadmium and zinc were toxic at 10⁻² M in media containing 0.01 to 1.0 mg of C liter⁻¹. At C concentrations greater than 1.0 mg liter⁻¹, the inhibition of glucose degradation and carbon assimilation was observed at 10⁻³ M Cd and Zn. The toxicity of mercury seemed to be independent of the C concentration. Results of this study showed that the nutritional state of an organism may have a profound effect on its sensitivity to metals. Metals taken up by an energy-driven transport system may be less toxic under conditions of C starvation. The C concentration should be taken into account when evaluating results from toxicity studies, especially as most microorganisms in nature live under energy-limited conditions.     

Direct Determination of Carbon and Nitrogen Contents of Natural Bacterial Assemblages in Marine Environments

In order to better estimate bacterial biomass in marine environments, we developed a novel technique for direct measurement of carbon and nitrogen contents of natural bacterial assemblages. Bacterial cells were separated from phytoplankton and detritus with glass fiber and membrane filters (pore size, 0.8 μm) and then concentrated by tangential flow filtration. The concentrate was used for the determination of amounts of organic carbon and nitrogen by a high-temperature catalytic oxidation method, and after it was stained with 4′,6-diamidino-2-phenylindole, cell abundance was determined by epifluorescence microscopy. We found that the average contents of carbon and nitrogen for oceanic bacterial assemblages were 12.4 ± 6.3 and 2.1 ± 1.1 fg cell⁻¹ (mean ± standard deviation; n = 6), respectively. Corresponding values for coastal bacterial assemblages were 30.2 ± 12.3 fg of C cell⁻¹ and 5.8 ± 1.5 fg of N cell⁻¹ (n = 5), significantly higher than those for oceanic bacteria (two-tailed Student’s t test; P < 0.03). There was no significant difference (P > 0.2) in the bacterial C:N ratio (atom atom⁻¹) between oceanic (6.8 ± 1.2) and coastal (5.9 ± 1.1) assemblages. Our estimates support the previous proposition that bacteria contribute substantially to total biomass in marine environments, but they also suggest that the use of a single conversion factor for diverse marine environments can lead to large errors in assessing the role of bacteria in food webs and biogeochemical cycles. The use of a factor, 20 fg of C cell⁻¹, which has been widely adopted in recent studies may result in the overestimation (by as much as 330%) of bacterial biomass in open oceans and in the underestimation (by as much as 40%) of bacterial biomass in coastal environments.     

Sea Ice Microbial Communities: Distribution, Abundance, and Diversity of Ice Bacteria in McMurdo Sound, Antarctica, in 1980

An abundant and diverse bacterial community was found within brine channels of annual sea ice and at the ice-seawater interface in McMurdo Sound, Antarctica, in 1980. The mean bacterial standing crop was 1.4 × 10¹¹ cells m⁻² (9.8 mg of C m⁻²); bacterial concentrations as high as 1.02 × 10¹² cells m⁻³ were observed in ice core melt water. Vertical profiles of ice cores 1.3 to 2.5 m long showed that 47% of the bacterial numbers and 93% of the bacterial biomass were located in the bottom 20 cm of sea ice. Ice bacterial biomass concentration was more than 10 times higher than bacterioplankton from the water column. Scanning electron micrographs showed a variety of morphologically distinct cell types, including coccoid, rod, fusiform, filamentous, and prosthecate forms; dividing cells were commonly observed. Approximately 70% of the ice bacteria were free-living, whereas 30% were attached to either living algal cells or detritus. Interactions between ice bacteria and microalgae were suggested by a positive correlation between bacterial numbers and chlorophyll a content of the ice. Scanning and transmission electron microscopy revealed a close physical association between epibacteria and a dominant ice alga of the genus Amphiprora. We propose that sea ice microbial communities are not only sources of primary production but also sources of secondary microbial production in polar ecosystems. Furthermore, we propose that a detrital food web may be associated with polar sea ice.     

Microbial Diversity in Maras Salterns, a Hypersaline Environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 × 10⁶ to 3 × 10⁶ cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon “Haloquadra walsbyi,” although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the γ-proteobacterium “Pseudomonas halophila” DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the “P. halophila” cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

Simultaneous Transport of Two Bacterial Strains in Intact Cores from Oyster, Virginia: Biological Effects and Numerical Modeling

The transport characteristics of two adhesion-deficient, indigenous groundwater strains, Comamonas sp. strain DA001 and Erwinia herbicola OYS2-A, were studied by using intact sediment cores (7 by 50 cm) from Oyster, Va. Both strains are gram-negative rods (1.10 by 0.56 and 1.56 by 0.46 μm, respectively) with strongly hydrophilic membranes and a slightly negative surface charge. The two strains exhibited markedly different behaviors when they were transported through granular porous sediment. To eliminate any effects of physical and chemical heterogeneity on bacterial transport and thus isolate the biological effect, the two strains were simultaneously injected into the same core. DA001 cells were metabolically labeled with ³⁵S and tagged with a vital fluorescent stain, while OYS2-A cells were metabolically labeled with ¹⁴C. The fast decay of ³⁵S allowed deconvolution of the two isotopes (and therefore the two strains). Dramatic differences in the transport behaviors were observed. The breakthrough of DA001 and the breakthrough of OYS2-A both occurred before the breakthrough of a conservative tracer (termed differential advection), with effluent recoveries of 55 and 30%, respectively. The retained bacterial concentration of OYS2-A in the sediment was twofold higher than that of DA001. Among the cell properties analyzed, the statistically significant differences between the two strains were cell length and diameter. The shorter, larger-diameter DA001 cells displayed a higher effluent recovery than the longer, smaller-diameter OYS2-A cells. CXTFIT modeling results indicated that compared to the DA001 cells, the OYS2-A cells experienced lower pore velocity, higher porosity, a higher attachment rate, and a lower detachment rate. All these factors may contribute to the observed differences in transport.     

Glycine Betaine, Carnitine, and Choline Enhance Salinity Tolerance and Prevent the Accumulation of Sodium to a Level Inhibiting Growth of Tetragenococcus halophila

Natural-abundance ¹³C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na⁺ content and confer a much higher salt tolerance to T. halophila.     

Characterization of the Polyurethanolytic Activity of Two Alicycliphilus sp. Strains Able To Degrade Polyurethane and N-Methylpyrrolidone

Two bacterial strains (BQ1 and BQ8) were isolated from decomposed soft foam. These were selected for their capacity to grow in a minimal medium (MM) supplemented with a commercial surface-coating polyurethane (PU) (Hydroform) as the carbon source (MM-PUh). Both bacterial strains were identified as Alicycliphilus sp. by comparative 16S rRNA gene sequence analysis. Growth in MM-PUh showed hyperbolic behavior, with BQ1 producing higher maximum growth (17.8 ± 0.6 mg·ml⁻¹) than BQ8 (14.0 ± 0.6 mg·ml⁻¹) after 100 h of culture. Nuclear magnetic resonance, Fourier transform infrared (IR) spectroscopy, and gas chromatography-mass spectrometry analyses of Hydroform showed that it was a polyester PU type which also contained N-methylpyrrolidone (NMP) as an additive. Alicycliphilus sp. utilizes NMP during the first stage of growth and was able to use it as the sole carbon and nitrogen source, with calculated K_s values of about 8 mg·ml⁻¹. Enzymatic activities related to PU degradation (esterase, protease, and urease activities) were tested by using differential media and activity assays in cell-free supernatants of bacterial cultures in MM-PUh. Induction of esterase activity in inoculated MM-PUh, but not that of protease or urease activities, was observed at 12 h of culture. Esterase activity reached its maximum at 18 h and was maintained at 50% of its maximal activity until the end of the analysis (120 h). The capacity of Alicycliphilus sp. to degrade PU was demonstrated by changes in the PU IR spectrum and by the numerous holes produced in solid PU observed by scanning electron microscopy after bacterial culture. Changes in the PU IR spectra indicate that an esterase activity is involved in PU degradation.