Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

Effect of growth on surface charge and hydrophobicity of Staphylococcus aureus

Modifications in the surface charge and hydrophobicity of Staphylococcus aureus Oxford during growth were studied by analysing electrophoretic mobility and adherence to hydrocarbons (hexadecane), respectively. Bacterial concentration had no effect upon the measurements. Both surface charge and hydrophobicity varied during the exponential phase of growth (1 to 4 h): surface charge decreased significantly (p < 0.001), while hydrophobicity increased (p < 0.001). In the stationary phase (4 to 9 h), the surface charge increased significantly (p < 0.001), whereas hydrophobicity showed no change. Cationized ferritin decreased the surface charge and had no effect on hydrophobicity. These results suggest that in S. aureus, different structures could be responsible for their surface charge and hydrophobic properties.     

Production and Properties of a Surface-Active Lipopeptide Produced by a New Marine Brevibacterium luteolum Strain

Microbial-derived surfactants are molecules of great interest due to their environmentally friendly nature and low toxicity; however, their production cost is not competitive when compared to synthetics. Marine microorganisms are exposed to extremes of pressure, temperature, and salinity; hence, they can produce stable compounds under such conditions that are useful for industrial applications. A screening program to select marine bacteria able to produce biosurfactant using low-cost substrates (mineral oil, sucrose, soybean oil, and glycerol) was conducted. The selected bacterial strain showed potential to synthesize biosurfactants using mineral oil as carbon source and was identified as Brevibacterium luteolum. The surface-active compound reduced the surface tension of water to 27 mN m^?1 and the interfacial tension (water/hexadecane) to 0.84 mN m^?1 and showed a critical micelle concentration of 40 mg L^?1. The biosurfactant was stable over a range of temperature, pH, and salt concentration and the emulsification index (E₂₄) with different hydrocarbons ranging from 60 to 79 %. Structural characterization revealed that the biosurfactant has a lipopeptide nature. Sand washing removed 83 % of crude oil demonstrating the potential of the biosurfactants (BS) for bioremediation purposes. The new marine B. luteolum strain showed potential to produce high surface-active and stable molecule using a low-cost substrate.     

活性炭孔径和比表面积对TiO2/AC光催化性能的影响

采用“同步物理-化学活化法”二次活化商品活性炭, 制备不同孔径和比表面积的系列活性炭(AC)载体, 以偏钛酸为钛源, 利用均匀沉淀法制备TiO2负载型光催化剂(TiO2/AC), 用氮吸附、XRD、SEM等方法表征, 研究活性炭的孔径和比表面积对TiO2/AC性能的影响; 并通过降解水溶液中的亚甲基蓝(MB)研究TiO2/AC光催化氧化特性, 考察催化剂投加量、不同MB浓度等因素对光催化氧化的影响. 结果表明, 负载的TiO2粒子粒径为12-20 nm, 活性炭的比表面积大、平均孔径大有利于TiO2的均匀分散, 阻止TiO2晶粒生长, 有利于充分发挥TiO2小尺度效应; 另外, 活性炭吸附和TiO2光解的协同效应使TiO2/AC光催化剂对MB降解的效率显著提高. 动力学研究表明, TiO2/AC光催化降解MB反应符合表观一级动力学特征.     

Atomic force microscopy measurement of heterogeneity in bacterial surface hydrophobicity

The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.     

Bacterial adhesion to glass and metal-oxide surfaces

Metal oxides can increase the adhesion of negatively-charged bacteria to surfaces primarily due to their positive charge. However, the hydrophobicity of a metal-oxide surface can also increase adhesion of bacteria. In order to understand the relative contribution of charge and hydrophobicity to bacterial adhesion, we measured the adhesion of 8 strains of bacteria, under conditions of low and high-ionic strength (1 and 100 mM, respectively) to 11 different surfaces and examined adhesion as a function of charge, hydrophobicity (water contact angle) and surface energy. Inorganic surfaces included three uncoated glass surfaces and eight metal-oxide thin films prepared on the upper (non-tin-exposed) side of float glass by chemical vapor deposition. The Gram-negative bacteria differed in lengths of lipopolysaccharides on their outer surface (three Escherichia coli strains), the amounts of exopolysaccharides (two Pseudomonas aeruginosa strains), and their known relative adhesion to sand grains (two Burkholderia cepacia strains). One Gram positive bacterium was also used that had a lower adhesion to glass than these other bacteria (Bacillus subtilis). For all eight bacteria, there was a consistent increase in adhesion between with the type of inorganic surface in the order: float glass exposed to tin (coded here as Si-Sn), glass microscope slide (Si-m), uncoated air-side float glass surface (Si-a), followed by thin films of (Co(1-y-z)Fe(y)Cr(z))3O4, Ti/Fe/O, TiO2, SnO2, SnO2:F, SnO2:Sb, A1(2)O3, and Fe2O3 (the colon indicates metal doping, a slash indicates that the metal is a major component, while the dash is used to distinguish surfaces). Increasing the ionic strength from 1 to 100 mM increased adhesion by a factor of 2.0 +/- 0.6 (73% of the sample results were within the 95% CI) showing electrostatic charge was important in adhesion. However, adhesion was not significantly correlated with bacterial charge and contact angle. Adhesion (A) of the eight strains was significantly (P < 10(-25)) correlated with total adhesion free energy (U) between the bacteria and surface (A = 2162e(-1.8U)).Although the correlation was significant, agreement between the model and data was poor for the low energy surfaces (R2 = 0.68), indicating that better models or additional methods to characterize bacteria and surfaces are still needed to more accurately describe initial bacterial adhesion to inorganic surfaces.     

Bio-detoxification Bacteria Isolated from Dye-Polluted Soils Promote Lactic Acid Production from Ammonia Pretreated Corn Stover

Agro-stovers are the most abundant substrates for producing lactic acid, which has great potential application in the production of biodegradable and biocompatible polylactic acid polymers. However, chemical pretreatments on agro-stovers generate inhibitors that repress the subsequent lactic acid fermentation. In this study, three bacterial strains (Enterococcus faecalis B101, Acinetobacter calcoaceticus C1, and Pseudomonas aeruginosa CS) isolated from dye-polluted soils could utilize phenolic inhibitor mimics (vanillin, 4- hydroxybenzaldehyde, or syringaldehyde) from alkaline pretreated corn stovers as a sole carbon source. Lactic acid titer increased from 27.42 g/L (Bacillus coagulans LA204 alone) to 44.76 g/L (CS and LA204) using 50 g/L glucose with 1 g/L 4-hydroxybenzaldehyde added. Lactic acid production from 50 g/L ammonia pretreated corn stover was increased nearly twofold by inoculating phenolic degradation bacteria and lactic acid bacteria (C1& Lactobacillus pentosus FL0421). In the control (FL0421 alone), only 16.98 g/L of lactic acid was produced. The isolated and identified strains degraded the phenolic compounds and increased the lactic acid production from glucose and ammonia pretreated corn stover. These characteristics of the strains support industrial application with efficient in situ detoxification of phenolic compounds during lactic acid production from agro-stovers using simultaneous saccharification and fermentation (SSF).

     

Graphite-like carbon deposited anatase TiO<Subscript>2</Subscript> single crystals as efficient visible-light photocatalysts

Graphite-like carbon deposited single-crystal anatase TiO₂ with exposed {001} facets was fabricated through a two-step solvothermal process by using glucose as carbon source. The physicochemical properties of the as-prepared samples were investigated by X-ray diffraction, Brunauer-Emmett-Teller, transmission electron microscopy, Raman, UV–vis diffuse reflectance spectra, electrochemical impedance spectroscopy and surface photovoltage spectroscopy. These results demonstrated that graphite-like carbon layers were deposited on the surface of TiO₂ single-crystal nanosheets with exposed highly reactive {001} facets via the dehydration of glucose during the process of hydrothermal treatment. The loading of the graphite-like carbon layers could effectively extend the light absorption edge of the single-crystal anatase TiO₂ nanosheets to visible light region and accelerate the separation of photo-generated electrons and holes, contributing an excellent visible-light driven photocatalytic performance to the graphite-like carbon deposited single-crystal anatase TiO₂ nanosheets for the degradation of methyl orange.     

Diversity of the surface properties of Lactococci and consequences on adhesion to food components

Bacteria possess surface properties, related to their charge, hydrophobicity and Lewis acid/base characteristics, that are involved in the attachment processes of microorganisms to surfaces. Fermentation bulks and food matrixes are complex heterogeneous media containing various components with different physicochemical characteristics. The aim of the present study was to investigate whether (i) bacteria present in a food matrix, interacted physicochemically at their surface level with the other constituents and (ii) the diversity of bacterial surface properties could result in a diversity of microbial adhesion to components and thus in a diversity of tolerance to toxic compounds. The surface properties of 20 lactic acid bacteria were characterized by the MATS method showing their relatively hydrophilic and various basic characteristics. The results obtained from a set of representative strains showed that (i) the strains with higher affinity for apolar solvents adsorbed more to lipids and hydrophobic compounds, (ii) the more the strains adsorbed to a toxic solvent, the less they were tolerant to this solvent. A diversity of bacterial surface properties was observed for the strains in the same species showing the importance of choosing bacteria according to their surface properties in function of technological objectives.     

Diversity of the surface properties of Lactococci and consequences on adhesion to food components

Bacteria possess surface properties, related to their charge, hydrophobicity and Lewis acid/base characteristics, that are involved in the attachment processes of microorganisms to surfaces. Fermentation bulks and food matrixes are complex heterogeneous media containing various components with different physicochemical characteristics. The aim of the present study was to investigate whether (i) bacteria present in a food matrix, interacted physicochemically at their surface level with the other constituents and (ii) the diversity of bacterial surface properties could result in a diversity of microbial adhesion to components and thus in a diversity of tolerance to toxic compounds. The surface properties of 20 lactic acid bacteria were characterized by the MATS method showing their relatively hydrophilic and various basic characteristics. The results obtained from a set of representative strains showed that (i) the strains with higher affinity for apolar solvents adsorbed more to lipids and hydrophobic compounds, (ii) the more the strains adsorbed to a toxic solvent, the less they were tolerant to this solvent. A diversity of bacterial surface properties was observed for the strains in the same species showing the importance of choosing bacteria according to their surface properties in function of technological objectives.     

双孔道管状有序介孔炭对维生素B12的吸附缓释性能

通过纳米铸型法,以硅基介孔分子筛SBA-15为模板,糠醇为碳源,草酸作为聚合催化剂合成了具有双孔道管状有序介孔炭CMK-5.利用粉末X射线衍射(XRD)、扫描电子显微镜(SEM)和氮气物理吸附法等对其结构进行了表征.结果表明合成的CMK-5具有有序结构,比表面积和孔容积分别高达1856 m2·g-1和2.10 cm3·g-1,在3.1和5.5nm具有集中的双孔分布.由于独特的双孔道结构特点,CMK-5在120 min内快速吸附维生素B12至平衡,吸附量高达943 mg·g-1,远高于商用活性炭.CMK-5吸附维生素B12后可以直接用于缓释,动态缓释浓度维持在～9 mg·L-1,适用于维生素B12分子在人体内的缓释.     

Role of lactobacillus cell surface hydrophobicity as probed by AFM in adhesion to surfaces at low and high ionic strength

The S-layer present at the outermost cell surface of some lactobacillus species is known to convey hydrophobicity to the lactobacillus cell surface. Yet, it is commonly found that adhesion of lactobacilli to solid substrata does not proceed according to expectations based on cell surface hydrophobicity. In this paper, the role of cell surface hydrophobicity of two lactobacillus strains with and without a surface layer protein (SLP) layer has been investigated with regard to their adhesion to hydrophobically or hydrophilically functionalized glass surfaces under well-defined flow conditions and in low and high ionic strength suspensions. Similarly, the interaction of the lactobacilli with similarly functionalized atomic force microscope (AFM) tips was measured. In a low ionic strength suspension, both lactobacillus strains show higher initial deposition rates to hydrophobic glass than to hydrophilic glass, whereas in a high ionic strength suspension no clear influence of cell surface hydrophobicity on adhesion is observed. Independent of ionic strength, however, AFM detects stronger interaction forces when both bacteria and tip are hydrophobic or hydrophilic than when bacteria and tip have opposite hydrophobicities. This suggest that the interaction develops in a different way when a bacterium is forced into contact with the tip surface, like in AFM, as compared with contacts developing between a cell surface and a macroscopic substratum under flow. In addition, the distance dependence of the total Gibbs energy of interaction could only be qualitatively correlated with bacterial deposition and desorption in the parallel plate flow chamber.     

Bacteria attachment to surfaces--AFM force spectroscopy and physicochemical analyses

Understanding bacterial adhesion to surfaces requires knowledge of the forces that govern bacterial-surface interactions. Biofilm formation on stainless steel 316 (SS316) by three bacterial species was investigated by examining surface force interaction between the cells and metal surface using atomic force microscopy (AFM). Bacterial-metal adhesion force was quantified at different surface delay time from 0 to 60s using AFM tip coated with three different bacterial species: Gram-negative Massilia timonae and Pseudomonas aeruginosa, and Gram-positive Bacillus subtilis. The results revealed that bacterial adhesion forces on SS316 surface by Gram-negative bacteria is higher (8.53±1.40 nN and 7.88±0.94 nN) when compared to Gram-positive bacteria (1.44±0.21 nN). Physicochemical analysis on bacterial surface properties also revealed that M. timonae and P. aeruginosa showed higher hydrophobicity and surface charges than B. subtilis along with the capability of producing extracellular polymeric substances (EPS). The higher hydrophobicity, surface charges, and greater propensity to form EPS by M. timonae and P. aeruginosa led to high adhesive force on the metal surface.     

Evaluation of Cashew Apple Juice for Surfactin Production by Bacillus subtilis LAMI008

Bacillus subtilis LAMI008 strain isolated from the tank of Chlorination at the Wastewater Treatment Plant on Campus do Pici in Federal University of Ceará, Brazil has been screened for surfactin production in mineral medium containing clarified cashew apple juice (MM-CAJC). Results were compared with the ones obtained using mineral medium with glucose PA as carbon source. The influence on growth and surfactin production of culture medium supplementation with yeast extract was also studied. The substrate concentration analysis indicated that B. subtilis LAMI008 was able to degrade all carbon sources studied and produce biosurfactant. The highest reduction in surface tension was achieved with the fermentation of MM-CAJC, supplemented with yeast extract, which decreased from 58.95?±?0.10 to 38.10?±?0.81 dyn cm^?1. The biosurfactant produced was capable of emulsifying kerosene, achieving an emulsification index of 65%. Surfactin concentration of 3.5 mg L^?1 was obtained when MM-CAJC, supplemented with yeast extract, was used, thus indicating that it is feasible to produce surfactin from clarified cashew apple juice, a renewable and low-cost carbon source.     

Enhanced Biodegradation of Alkane Hydrocarbons and Crude Oil by Mixed Strains and Bacterial Community Analysis

In this study, two strains, Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01, were isolated from soil samples polluted by crude oil at Bohai offshore. The former one could degrade alkane hydrocarbons (crude oil and diesel, 1:4 (v/v)) and crude oil efficiently; the latter one failed to grow on alkane hydrocarbons but could produce rhamnolipid (a biosurfactant) with glycerol as sole carbon source. Compared with pure culture, mixed culture of the two strains showed higher capability in degrading alkane hydrocarbons and crude oil of which degradation rate were increased from 89.35 and 74.32?±?4.09 to 97.41 and 87.29?±?2.41 %, respectively. In the mixed culture, Acinetobacter sp. XM-02 grew fast with sufficient carbon source and produced intermediates which were subsequently utilized for the growth of Pseudomonas sp. XM-01 and then, rhamnolipid was produced by Pseudomonas sp. XM-01. Till the end of the process, Acinetobacter sp. XM-02 was inhibited by the rapid growth of Pseudomonas sp. XM-01. In addition, alkane hydrocarbon degradation rate of the mixed culture increased by 8.06 to 97.41 % compared with 87.29 % of the pure culture. The surface tension of medium dropping from 73.2?×?10^?3 to 28.6?×?10^?3 N/m. Based on newly found cooperation between the degrader and the coworking strain, rational investigations and optimal strategies to alkane hydrocarbons biodegradation were utilized for enhancing crude oil biodegradation.     

结构可切换型聚合物刷的构建及表面性能

合成了结构可切换型甲基丙烯酸酯季铵盐(CBMA-1C2). 在聚丙烯(PP)片基表面光接枝构建CBMA-1C2聚合物刷, 其在碱性水溶液中可水解形成两性离子聚合物刷PCBMA. 用蛋白质吸附及血小板黏附实验评价改性表面亲/疏水性及表面电荷对生物分子与材料表面之间相互作用的影响. 结果发现, 与未改性PP片基相比, 聚合物PCBMA-1C2改性表面水解前后均具有优异的亲水性能, 由于聚合物PCBMA-1C2水解前后表面电荷不同, 对生物分子与改性PP表面的相互作用表现出明显差异. 亲水性好、 两性离子结构的聚合物PCBMA表面表现出对蛋白吸附和血小板黏附的良好抑制作用.     

Poly-β-hydroxybutyrate and Exopolysaccharide Biosynthesis by Bacterial Isolates from Pigeonpea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp] Root Nodules

The bacterial strains that are able to produce biopolymers that are applied in industrial sectors present a source of renewable resources. Some microorganisms are already applied at several industrial sectors, but the prospecting of new microbes must bring microorganisms that are feasible to produce interesting biopolymers more efficiently and in cheaper conditions. Among the biopolymers applied industrially, polyhydroxybutyrate (PHB) and exopolysaccharides (EPS) stand out because of its applications, mainly in biodegradable plastic production and in food industry, respectively. In this context, the capacity of bacteria isolated from pigeonpea root nodules to produce EPS and PHB was evaluated, as well as the cultural characterization of these isolates. Among the 38 isolates evaluated, the majority presented fast growth and ability to acidify the culture media. Regarding the biopolymer production, five isolates produced more than 10 mg PHB per liter of culture medium. Six EPS producing bacteria achieved more than 200 mg EPS per liter of culture medium. Evaluating different carbon sources, the PHB productivity of the isolate 24.6b reached 69% of cell dry weight when cultured with starch as sole carbon source, and the isolate 8.1c synthesized 53% PHB in dry cell biomass and more than 1.3 g L⁻¹ of EPS when grown using xylose as sole carbon source.     

The phenol efficient degradation using co-culture coupled with enhanced electricity generation capability

The co-culture of strain Citrobacter sp. RDC and Geobacter sulfurreducens PCA was used in this study and it was found that the co-culture using 200 mg/L phenol as carbon source exhibited higher maximum current density than using the single strain RDC and G. sulfurreducens PCA, respectively. Meanwhile, the co-culture was used to generate electricity by degrading phenol with the current density of 699.07 μA/cm² by using 200 mg/L phenol as the sole carbon source, which was higher than that only using G. sulfurreducens PCA (236.20 μA/cm²). Especially, the degradation efficiency of 200 mg/L phenol by co-culture can reach 55.16 % within 36 h being 4.16-fold higher than the single strain G. sulfurreducens PCA. Furthermore, the component ratio of two strains was optimized for increasing electricity generation using 500 mg/L phenol as carbon source. The maximum current density was 501.54 μA/cm² under the ratio of 3 : 1 for strain RDC to G. sulfurreducens PCA. These results highlight that phenol is good carbon source for co-culture to produce electricity. The co-culture system provides a promising application pathway for phenol degradation treatment coupled with electricity generation in the future.     

Influence of the Carbon Source on Gordonia alkanivorans Strain 1B Resistance to 2-Hydroxybiphenyl Toxicity

The viability of bacteria plays a critical role in the enhancement of fossil fuels biodesulfurization efficiency since cells are exposed to toxic compounds such as 2-hydroxybiphenyl (2-HBP), the end product of dibenzothiophene (DBT) biodesulfurization. The goal of this work was to study the influence of the carbon source on the resistance of Gordonia alkanivorans strain 1B to 2-HBP. The physiological response of this bacterium, pre-grown in glucose or fructose, to 2-HBP was evaluated using two approaches: a growth inhibition toxicity test and flow cytometry. The results obtained from the growth inhibition bioassays showed that the carbon source has an influence on the sensitivity of strain 1B growing cells to 2-HBP. The highest IC₅₀ value was obtained for the assay using fructose as carbon source in both inoculum growth and test medium (IC₅₀-48 h?=?0.464 mM). Relatively to the evaluation of 2-HBP effect on the physiological state of resting cells by flow cytometry, the results showed that concentrations of 2-HBP >1 mM generated significant loss of cell viability. The higher the 2-HBP concentration, the higher the toxicity effect on cells and the faster the loss of cell viability. In overall, the flow cytometry results highlighted that strain 1B resting cells grown in glucose-SO₄ or glucose-DBT are physiologically less resistant to 2-HBP than resting cells grown in fructose-SO₄ or fructose-DBT, respectively.     

Effects of carbon source on expression of alcohol oxidase activity and on morphologic pattern of YR-1 strain, a filamentous fungus isolated from petroleum-contaminated soils

Soluble alcohol oxidase (AO) activity was detected in the supernatant fraction of a high-speed centrifugation procedure after ballistic cellular homogenization to break the mycelium from a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing different carbon sources, including alcohols and hydrocarbons but not in glucose. In previous work, zymogram analysis conducted with crude extracts from aerobic mycelium of YR-1 strain indicated the existence of two AO enzymes originally named AO-1 and AO-2. In the present study, we were able to separate the AO-1 band into two bands depending on culture conditions, carbon source, and polyacrylamide gel electrophoresis (PAGE) separation conditions; the enzyme activity pattern in zymograms from cell-free extracts exhibited three different bands after native PAGE. New nomenclature was used for upper bands AO-1 and AO-2 and lower band AO-3, respectively. The expression of AO activity was studied in the absence of glucose in the culture media and in the presence of hydrocarbons or petroleum as sole carbon source, suggesting that AO expression could be subjected to two regulatory possibilities: carbon catabolite regulation by glucose and induction by hydrocarbons. The possibility of catabolic inhibition of AO by glucose in the active enzyme was also tested, and the results confirm that this kind of regulatory mechanism is not present in AO activity.