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Applied Microbiology and Biotechnology (v.64, #3)
Micro total analysis system (μ-TAS) in biotechnology by S. J. Lee; S. Y. Lee (pp. 289-299).
Nanobiotechnology raises fascinating possibilities for new analytical assays in various fields such as bioelectronic assembly, biomechanics and sampling techniques, as well as in chips or micromachined devices. Recently, nanotechnology has greatly impacted biotechnological research with its potential applications in smart devices that can operate at the level of molecular manipulation. Micro total analysis system (μ-TAS) offers the potential for highly efficient, simultaneous analysis of a large number of biologically important molecules in genomic, proteomic and metabolic studies. This review aims to describe the present state-of-the-art of microsystems for use in biotechnological research, medicine and diagnostics.
Chirality of pollutants—effects on metabolism and fate by T. A. Müller; H.-P. E. Kohler (pp. 300-316).
In most cases, enantiomers of chiral compounds behave differently in biochemical processes. Therefore, the effects and the environmental fate of the enantiomers of chiral pollutants need to be investigated separately. In this review, the different fates of the enantiomers of chiral phenoxyalkanoic acid herbicides, acetamides, organochlorines, and linear alkylbenzenesulfonates are discussed. The focus lies on biological degradation, which may be enantioselective, in contrast to non-biotic conversions. The data show that it is difficult to predict which enantiomer may be enriched and that accumulation of an enantiomer is dependent on the environmental system, the species, and the organ. Racemization and enantiomerization processes occur and make interpretation of the data even more complex. Enantioselective degradation implies that the enzymes involved in the conversion of such compounds are able to differentiate between the enantiomers. “Enzyme pairs” have evolved which exhibit almost identical overall folding. Only subtle differences in their active site determine their enantioselectivities. At the other extreme, there are examples of non-homologous “enzyme pairs” that have developed through convergent evolution to enantioselectively turn over the enantiomers of a chiral compound. For a better understanding of enantioselective reactions, more detailed studies of enzymes involved in enantioselective degradation need to be performed.
Microbial P450 enzymes in biotechnology by V. B. Urlacher; S. Lutz-Wahl; R. D. Schmid (pp. 317-325).
Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes since they allow the introduction of oxygen into non-activated carbon atoms in a sterically and optically selective manner that is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. Surprisingly, cytochrome P450 monooxygenases have scarcely been studied for use in biooxidations, although they are one of the largest known superfamilies of enzyme proteins. Their gene sequences have been identified in various organisms such as humans, bacteria, algae, fungi, and plants. The reactions catalyzed by P450s are quite diverse and range from biosynthetic pathways (e.g. those of animal hormones and secondary plant metabolites) to the activation or biodegradation of hydrophobic xenobiotic compounds (e.g. those of various drugs in the liver of higher animals). From a practical point of view, the great potential of P450s is limited by their functional complexity, low activity, and limited stability. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways that could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes, and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are both covered in this review.
Reutericyclin: biological activity, mode of action, and potential applications by M. G. Gänzle (pp. 326-332).
Reutericyclin is an inhibitory compound produced by sourdough isolates of Lactobacillus reuteri that is structurally but not functionally related to naturally occurring tetramic acids. It is bacteriostatic or bactericidal to gram-positive bacteria based on its activity as a proton-ionophore, and a broad range of food-related spoilage organisms and pathogens is inhibited by reutericyclin. Gram-negative bacteria are resistant to reutericyclin because of the barrier properties of their outer membrane, and resistance of beer-spoiling lactobacilli towards hop bitter acids provides cross-protection to reutericyclin. Remarkably, reutericyclin-producing strains were shown to persist for a period of 10 years in an industrial sourdough fermentation, and reutericyclin was shown to be produced in concentrations active against competitors during growth of L. reuteri in sourdough. Based on the known properties of reutericyclin and L. reuteri, reutericyclin-producing strains may have applications in the biopreservation of foods. Furthermore, these strains were shown to colonize reconstituted lactobacilli-free mice at high levels. Therefore, they could serve as a suitable model system to evaluate a possible impact of antimicrobial compounds on the intestinal microflora of humans and animals.
Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for d-mannitol formation in a whole-cell biotransformation by B. Kaup; S. Bringer-Meyer; H. Sahm (pp. 333-339).
A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of d-fructose to d-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce d-mannitol from d-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form d-mannitol from d-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up d-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM d-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD+-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y D-mannitol/D-fructosewas 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.
Evaluation of different organic phases for water-in-oil xanthan fermentation by S. G. Kuttuva; A. S. Restrepo; L.-K. Ju (pp. 340-345).
Water-in-oil (W/O) fermentation technology has the potential for overcoming the problems related with high broth viscosity in xanthan fermentations. By dispersing the aqueous broth in a continuous organic phase, the broth-thickening mechanisms are confined within the aqueous droplets without significantly increasing the overall viscosity. In this study, xanthan fermentations were made with perfluorocarbon (PFC) or vegetable oil as the organic phase. The results were compared with those obtained previously using n-hexadecane as the organic phase, to evaluate the effects of various properties. PFC provided easy phase separation at the end of fermentation but required higher power input for agitation, a direct result of its high density. The aqueous droplets formed were large (400–450 μm), limiting the cell concentration employable due to the occurrence of oxygen starvation in the inner core. One main advantage of using vegetable oil was its low cost. In addition, vegetable oil provided much finer droplets (<120 μm) and produced high xanthan concentrations (>100 g l−1). However, complete phase separation for product recovery was difficult to achieve. Fermentations in both organic phases were terminated by the occurrence of phase inversion to highly viscous O/W dispersions at aqueous-phase volume fractions of 0.53–0.56. The initial fraction was 0.3 but changed due to base addition for pH adjustment and nutrient addition for prolonged production.
Natural and recombinant fungal laccases for paper pulp bleaching by C. Sigoillot; E. Record; V. Belle; J. L. Robert; A. Levasseur; P. J. Punt; C. A. M. J. J. van den Hondel; A. Fournel; J. C. Sigoillot; M. Asther (pp. 346-352).
Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l−1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (K m) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).
Improved xylanase production by Trichoderma reesei grown on l-arabinose and lactose or d-glucose mixtures by H. Xiong; O. Turunen; O. Pastinen; M. Leisola; N. von Weymarn (pp. 353-358).
Trichoderma reesei Rut C-30 was grown on eight different natural or rare aldopentoses as the main carbon source and on mixtures of an aldopentose with d-glucose or lactose. The fungal cells consumed all aldopentoses tested, except l-xylose and l-ribose. The highest total xylanase and cellulase activities were achieved when cells were grown on l-arabinose as the main carbon source. The total xylanase activity produced by cells grown on l-arabinose was even higher than that produced by cells grown on an equal amount of lactose. In co-metabolism of d-glucose (15 g l−1) and l-arabinose (5 g l−1), the total volumetric and specific xylanase productivities were improved (derepressed) approximately 23- and 18-fold, respectively, compared to a cultivation on only d-glucose (20 g l−1). In a similar experiment, in which cells were grown on a mixture of lactose and l-arabinose, the xylanase productivity was approximately doubled, compared to a cultivation on only lactose. The cellulase productivities, however, were not improved by the addition of l-arabinose. Compared with a typical industrial fungal enzyme production process with lactose as the main carbon source, better volumetric and specific xylanase productivities were achieved both on a lactose/arabinose mixture and on a glucose/arabinose mixture.
Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis by M. Kataoka; A.-R. G. Delacruz-Hidalgo; M. A. Akond; E. Sakuradani; K. Kita; S. Shimizu (pp. 359-366).
The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to d-pantoyl lactone.
Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis by M. Kataoka; A.-R. G. Delacruz-Hidalgo; M. A. Akond; E. Sakuradani; K. Kita; S. Shimizu (pp. 359-366).
The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to d-pantoyl lactone.
The effect of pfl gene knockout on the metabolism for optically pure d-lactate production by Escherichia coli by J. Zhu; K. Shimizu (pp. 367-375).
The effect of gene knockout on metabolism in the pflA−, pflB−, pflC−, and pflD− mutants of Escherichia coli was investigated. Batch cultivations of the pfl − mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA− and pflB− mutants, but not pflC− and pflD− mutants, produced large amounts of d-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD+ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA− and pflB− mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD+ ratio in pfl − mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.
Non-conventional yeasts as producers of polyhydroxyalkanoates—genetic engineering of Arxula adeninivorans by Y. Terentiev; U. Breuer; W. Babel; G. Kunze (pp. 376-381).
The non-conventional yeast Arxula adeninivorans was equipped with the genes phbA, phbB and phbC of the polyhydroxyalkanoate (PHA) biosynthetic pathway of Ralstonia eutropha, which encode β-ketothiolase, NADPH-linked acetoacetyl-CoA reductase and PHA synthase, respectively. Arxula strains transformed solely with the PHA synthase gene (phbC) were able to produce PHA. However, the maximum content of the polymer detected in these strains was just 0.003% poly-3-hydroxybutyrate (PHB) and 0.112% poly-3-hydroxyvalerate (PHV). The expression of all three genes (phbA, phbB, phbC) resulted in small increases in the PHA content of the transgenic Arxula cells. However, under controlled cultivation conditions with minimal medium and ethanol as the carbon source, the recombinant yeast was able to accumulate up to 2.2% PHV and 0.019% PHB. Possible reasons for these differences are discussed.
Gene replacement method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability by H. Yakhnin; P. Babitzke (pp. 382-386).
We describe a method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability. This method utilizes a chloramphenicol-resistant plasmid containing a temperature-sensitive (ts) replication origin. In this method, the target gene is first cloned into the ts vector and the recombinant plasmid is used to transform wild-type B. subtilis. The second step involves transformation of the resulting strain with a linear DNA fragment containing a second antibiotic resistance marker (tet) that disrupts the gene of interest. Selection for tetracycline resistance forces a double crossover between the chromosomal and fragment-borne copies of the gene, thereby replacing the wild-type gene in the chromosome with the disrupted allele. Cells survive even if the gene is essential by virtue of the complementing plasmid. Transformants are then grown at the non-permissive temperature for plasmid replication under various growth conditions. Isolation of chloramphenicol-sensitive colonies indicates that the gene is dispensable, whereas the inability to isolate chloramphenicol-sensitive colonies indicates that the gene is essential. The general utility of this method is demonstrated by allowing disruption of mtrA and trpE under conditions that render each gene non-essential, but not under growth conditions in which each gene is essential.
Degradation of 2-fluorophenol by the brown-rot fungus Gloeophyllum striatum: evidence for the involvement of extracellular Fenton chemistry by C. Kramer; G. Kreisel; K. Fahr; J. Käßbohrer; D. Schlosser (pp. 387-395).
Iron-containing liquid cultures of the brown-rot basidiomycete Gloeophyllum striatum degraded 2-fluorophenol. Two simultaneously appearing degradation products, 3-fluorocatechol and catechol, were identified by gas chromatography and mass spectrometry (GC-MS). Concomitantly, fluoride was produced at approximately 50% of the amount that theoretically could be achieved upon complete dehalogenation. Defluorination was strongly inhibited in the presence of either the hydroxyl radical scavenger mannitol or superoxide dismutase, as well as in the absence of iron. The addition of the natural iron chelator oxalate caused a clear but less extensive inhibition, whereas supplementation with the artificial iron chelator nitrilotriacetic acid increased fluoride production. Extracellular 2-fluorophenol degradation was evidenced by defluorination, observed upon addition of 2-fluorophenol to cell-free culture supernatants derived from iron-containing fungal cultures. Ultrafiltered culture supernatants oxidized methanol to formaldehyde, known as a product of the reaction of methanol with hydroxyl radical. In addition, G. striatum was found to produce metabolites extractable with ethyl acetate that are capable of reducing Fe3+. GC-MS analysis of such extracts revealed the presence of several compounds. The mass spectrum of a prominent peak matched those previously reported for 2,5-dimethoxyhydroquinone and 4,5-dimethoxycatechol, fungal metabolites implicated to drive hydroxyl radical production in Gloeophyllum. Taken together, these findings further support an extracellular Fenton-type mechanism operative during halophenol degradation by G. striatum.
Diversity of l-leucine catabolism in various microorganisms involved in dairy fermentations, and identification of the rate-controlling step in the formation of the potent flavour component 3-methylbutanal by B. A. Smit; W. J. M. Engels; J. T. M. Wouters; G. Smit (pp. 396-402).
Various microorganisms, belonging to the genera Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Propionibacterium, Brevibacterium, Corynebacterium and Arthrobacter, used in dairy fermentations such as cheese making, were analysed for their potential to convert leucine into flavour components, most notably 3-methylbutanal. A large variation between and within species was observed for various enzyme activities involved in the conversion pathway, e.g. transaminases, α-hydroxy acid dehydrogenase and α-keto acid decarboxylase. In particular, α-keto acid decarboxylase activity—leading to 3-methylbutanal—was found to be present in only two of the strains tested. It is proposed that this activity is rate-controlling in the conversion pathway leading to the flavour compound 3-methylbutanal.
Metabolite profiles of the biocontrol yeast Pichia anomala J121 grown under oxygen limitation by E. Fredlund; A. Broberg; M. E. Boysen; L. Kenne; J. Schnürer (pp. 403-409).
The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (a), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (a), but continued to be produced in (b). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (b). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (b) than in (a), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.
A thermodynamic interpretation of cell hydrophobicity in aerobic granulation by Y. Liu; S.-F. Yang; L. Qin; J.-H. Tay (pp. 410-415).
Aerobic granulation can be regarded as a microorganism-to-microorganism self-immobilization process, in which cell hydrophobicity could be a decisive parameter in determining the microorganism-to-microorganism interaction and structural compactness of aerobic granules. This study looked into the thermodynamic interpretation of cell hydrophobicity in aerobic granulation; and a model that correlates microbial interaction and relative cell hydrophobicity defined as the ratio of cell hydrophobicity over cell hydrophilicity was derived. This model describes how cell hydrophobic and hydrophilic interactions affect aerobic granulation and offers deep insights into the thermodynamic mechanisms of microbial aggregation. The model prediction was in good agreement with experimental data. Results showed that aerobic granulation was a function of cell hydrophobicity over cell hydrophilicity, i.e. a high cell hydrophobicity strongly favors microbial aggregation and results in a more compact structure.
Cerebrosides of Candida lipolytica yeast by J. Rupčić; V. Marić (pp. 416-420).
Candida lipolytica yeast was grown batchwise on glucose medium. Cerebrosides were isolated from the sphingolipid fraction of total lipids using column chromatography and separated into two compounds by high-performance thin-layer chromatography. Glucose was detected as the sole sugar constituent in cerebrosides. The fatty acid composition of cerebrosides was characterised by a predominance of saturated fatty acids and by a high proportion of fatty acids with 16 carbon atoms. The dominant fatty acid was h16:0. The principal long-chain base components of both cerebroside species were trihydroxy bases, 18- and 20-phytosphinosine. The unique characteristic of cerebrosides was the presence of a high proportion of sphingosine (one-fourth of the total long-chain bases), which is a common characteristic of mammalian sphingolipids and rarely occurs in yeast cerebrosides. The ceramide moiety profile of cerebrosides is similar to that of epidermal ceramides, which implies a possibility for their application in care cosmetics.
Effect of carbon monoxide, hydrogen and sulfate on thermophilic (55°C) hydrogenogenic carbon monoxide conversion in two anaerobic bioreactor sludges by J. Sipma; R. J. W. Meulepas; S. N. Parshina; A. J. M. Stams; G. Lettinga; P. N. L. Lens (pp. 421-428).
The conversion routes of carbon monoxide (CO) at 55°C by full-scale grown anaerobic sludges treating paper mill and distillery wastewater were elucidated. Inhibition experiments with 2-bromoethanesulfonate (BES) and vancomycin showed that CO conversion was performed by a hydrogenogenic population and that its products, i.e. hydrogen and CO2, were subsequently used by methanogens, homo-acetogens or sulfate reducers depending on the sludge source and inhibitors supplied. Direct methanogenic CO conversion occurred only at low CO concentrations [partial pressure of CO (P CO) <0.5 bar (1 bar=105 Pa)] with the paper mill sludge. The presence of hydrogen decreased the CO conversion rates, but did not prevent the depletion of CO to undetectable levels (<400 ppm). Both sludges showed interesting potential for hydrogen production from CO, especially since after 30 min exposure to 95°C, the production of CH4 at 55°C was negligible. The paper mill sludge was capable of sulfate reduction with hydrogen, tolerating and using high CO concentrations (P CO>1.6 bar), indicating that CO-rich synthesis gas can be used efficiently as an electron donor for biological sulfate reduction.
Survival of naphthalene-degrading Pseudomonas putida NCIB 9816-4 in naphthalene-amended soils: toxicity of naphthalene and its metabolites by W. Park; C. O. Jeon; H. Cadillo; C. DeRito; E. L. Madsen (pp. 429-435).
Survival of naphthalene-degrading Pseudomonas putida NCIB 9816-4 was measured in nonsterile soil samples (coal tar-contaminated and pristine) with and without added crystalline naphthalene over a period of 21 days. A 2–3 log decrease in cfu occurred in the presence, but not absence, of added naphthalene. We used aqueous suspensions of crystalline naphthalene to explore potential mechanisms of its toxicity on the test bacterium under aerobic conditions. Measurements of dissolved naphthalene in medium indicated that uptake by P. putida NCIB 9816-4 maintained naphthalene at concentrations well below saturation. Accumulation of catechol was documented by high-performance liquid chromatography and gas chromatography/mass spectrometry in the presence of 0.5% (w/v) naphthalene crystals. Transient catechol accumulation was highest when cells entered stationary phase. A decrease in catechol concentration correlated with the development of brown color in the medium. Brown pigment accumulation correlated with a decrease in viable cell counts. These results suggested that catechol, related compounds, and their condensation products can accumulate to toxic levels in stationary phase P. putida NCIB 9816-4 cells. We hypothesize that the same mechanism of toxicity may occur under the nutrient-limited conditions expected in soil.
