Plasmid-encoded enzymatic degradation of dimethoate

Dimethoate-degrading enzymatic activity in Bacillus licheniformis, Pseudomonas aeruginosa, Aeromonas hydrophila, Proteus mirabilis and Bacillus pumilus was found to be 6.4, 1.760, 4.09, 1.196 and 0.505 units/mg protein, respectively. The Escherichia coli C600 transconjugants of the isolated bacterial strains also exhibited dimethoate-degrading enzymatic activities. The cured derivatives did not show any decrease in the amount of dimethoate substrate and did not harbour plasmid as found in the original and transconjugant strains. Thus, the ability of enzymatic degradation of dimethoate was plasmid-mediated in B. licheniformis, Ps. aeruginosa, A. hydrophila, P. mirabilis and B. pumilus.     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

Degeneration of solventogenic Clostridium strains monitored by Fourier transform infrared spectroscopy of bacterial cells

Strain degeneration in solventogenic clostridia is a known problem in the technical acetone–butanol fermentation bioprocess, especially in the continuous process mode. Clostridial strain degeneration was studied by Fourier transform infrared (FT-IR) spectroscopy of the bacterial cells. Degenerative variant formation in two strains, Clostridium beijerinckii NCIMB 8052 and Clostridium species AA332, was detected spectroscopically. Colonies on solid media were sampled, or assayed directly in situ by IR microscopy. It has previously been shown that the distinctive acidogenic and solventogenic physiological phases of Clostridium acetobutylicum in liquid medium can be discriminated by FT-IR spectroscopy. This was confirmed here for C. beijerinckii NCIMB 8052. The proportion of degenerate cells in a mixed population in liquid medium could be quantified, as the spectral features change in different ways during the normal growth cycle of wild type organisms and degenerate variants in batch culture. This opens a new perspective for physiology-based process monitoring and control, especially of the continuous acetone–butanol fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 314–321. Received 06 October 2000/ Accepted in revised form 20 April 2001     

Isolation and partial purification of an antimicrobial agent from halotolerant alkaliphilic <Emphasis Type="Italic">Streptomyces aburaviensis</Emphasis> strain Kut-8

A halotolerant alkaliphilic actinomycete, Kut-8, was isolated from saline desert of Kutch, Western India. It has been identified as Streptomyces aburaviensis based on the chemotaxonomic characteristics, including cell wall constituents. Kut-8 is Gram-positive having a spiral sporophore with dark green and fluffy spore mass. It was able to grow with 15%, w/v NaCl with optimum being in the range of 5–10%. It grew optimally at pH 9 with slow growth at neutral pH. The cell wall contained l-diaminopimelic acid and no diagnostic sugars. It produced an antibiotic that selectively inhibited the growth of Gram-positive bacteria, with Bacillus subtilis being the most sensitive. Kut-8 secreted the antibiotic optimally during mid-stationary phase (on day 14 of growth in liquid culture). The crude antibiotic metabolites were separated by various solvent systems with hexane–methanol–water giving the best separation. The results of bioautographs revealed the presence of single active compound in the Kut-8 antibiotic filtrate. Partial purification of antibiotic metabolite by charcoal absorption and methanol extraction resulted in enhanced antimicrobial activity by 4.16-fold. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes from saline deserts have been explored and information on their antimicrobial potential is still scarce.     

Biosurfactant Facilitated Biodegradation of Quinalphos at High Concentrations by Pseudomonas aeruginosa Q10

Previous studies indicate that high concentration of pesticides and their associated toxic effects are high at their point source of application. Use of pesticide-degrading bacteria at point sources could augment degradation and thereby reduce toxic effects associated with pesticide persistence in soil. Quinalphos, an organophosphorus insecticide, though ranked “moderately hazardous” in the WHO's acute hazard ranking, still continues to be used extensively in developing countries. The presence of a chloride radical usually makes this pesticide sparingly soluble in water and hence difficult to degrade. The present study aimed to isolate autochthonous bacterial strains capable of utilizing quinalphos as a carbon source. Primary screening of pesticide-contaminated soil by enrichment culture and degradation analysis by UV-VIS spectrophotometry led to the isolation of 12 different bacterial isolates, of which three efficient isolates of Pseudomonas sp, Serratia sp, and Pseudomonas aeruginosa with degradation rate 86%, 82%, 94%, respectively, were selected. GC-MS studies with P.aeruginosa confirmed the formation of 2-hydroxy quinoxaline and phosphorothioic acid as a result of biodegradation. The present study succeeded in isolating autochthonous bacterial strains capable of utilizing high concentrations of quinalphos as a carbon source in a shorter incubation period. This strain also possessed biosurfactant-production ability, which makes quinalphos available to cells at higher concentrations.     

Antioxidant and antimicrobial actions of the clubmoss <Emphasis Type="Italic">Lycopodium clavatum</Emphasis> L.

Purpose of the present study was to evaluate antioxidant, antibacterial, antifungal, and antiviral activities of the petroleum ether, chloroform, ethyl acetate and methanol extracts as well as the alkaloid fraction of Lycopodium clavatum L. (LC) from Lycopodiaceae growing in Turkey. Antioxidant activity of the LC extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method at 0.2 mg/ml using microplate-reader assay. Antiviral assessment of LC extracts was evaluated towards the DNA virus Herpes simplex (HSV) and the RNA virus Parainfluenza (PI-3) using Madin-Darby Bovine Kidney (MDBK) and Vero cell lines. Antibacterial and antifungal activities of the extracts were tested against standard and isolated strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis as well as the fungi; Candida albicans and C. parapsilosis. All of the extracts possessed noteworthy activity against ATCC strain of S. aureus (4 μg/ml), while the LC extracts showed reasonable antifungal effect. On the other hand, we found that only the chloroform extract was active against HSV (16–8 μg/ml), while petroleum ether and alkaloid extracts inhibited potently PI-3 (16–4 μg/ml and 32–4 μg/ml, respectively). However, all of the extracts had insignificant antiradical effect on DPPH. In addition, we also analyzed the content of the alkaloid fraction of the plant by capillary gas chromatography-mass spectrometry (GC-MS) and identified lycopodine as the major alkaloid.     

Isolation and characterization of a <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strain capable of degrading zearalenone

The worldwide contamination of cereals, oilseeds, and other crops by mycotoxin-producing moulds is a significant problem. Mycotoxins have adverse effects on humans and animals that result in illnesses and economic losses. Reduction or elimination of mycotoxin contamination in food and feed is an important issue. This study aimed to screen soil bacteria for degradation of zearalenone (ZEN). A pure culture of strain CK1 isolated from soil samples showed most capable of degradation of ZEN. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, CK1 was identified as Bacillus licheniformis. Addition of 2 ppm of ZEN in Luria–Bertani (LB) medium, B. licheniformis CK1 decreased 95.8% of ZEN after 36 h of incubation. In ZEN-contaminated corn meal medium, B. licheniformis CK1 decreased more than 98% of ZEN after 36 h of incubation. In addition, B. licheniformis CK1 was non-hemolytic, non-enterotoxin producing, and displayed high levels of extracellular xylanase, cellulase, and protease activities. These findings suggest that B. licheniformis CK1 could be used to reduce the concentrations of ZEN and improve the digestibility of nutrients in feedstuffs simultaneously.     

Isolation of myo-Inositol from Solutions by Enzymatic Biotransformation

The transferase reaction between phospholipids and inositol catalyzed by phospholipase D was studied at interfaces in water–organic solvent systems. Optimum conditions were determined for phosphatidylinositol synthesis in heterogeneous water–organic solvent systems. Hydrophobic components (phospholipids) were readily separated from water-soluble products (alcohols) in systems with organic solvents. In the hexane–water system, addition of methanol (an alcohol substrate) to the reaction medium displaced myo-inositol from the molecule of phosphatidylinositol. myo-Inositol was isolated from the mixture of its isomers using a two-step transferase reaction catalyzed by phospholipase D.     

A halotolerant and thermotolerant <Emphasis Type="Italic">Bacillus</Emphasis> sp. degrades hydrocarbons and produces tensio-active emulsifying agent

A Bacillus sp. strain DHT, isolated from oil-contaminated soil, grew and produced biosurfactant when cultured in variety of substrate at salinities of up to 100 g l⁻¹ and temperatures up to 45°C. It was capable of utilizing crude oil, fuels, various pure alkanes and PAHs as a sole carbon and energy source across a wide range of temperature and salinity. Over the range evaluated, the degradation of hydrocarbon and biosurfactant production was not influenced by salinity (0–10% wv⁻¹) and temperature (30–45°C). The biosurfactant produced by the organism emulsified a range of hydrocarbons with hexadecane as the best substrate and toluene as the poorest. From 16S rDNA analysis, strain DHT was related to Bacillus licheniformis.     

Co-metabolic degradation of dimethoate by Raoultella sp. X1

A bacterium Raoultella sp. X1, based on its 16S rRNA gene sequence, was isolated. Characteristics regarding the bacterial morphology, physiology, and genetics were investigated with an electron microscopy and conventional microbiological techniques. Although the isolate grew and degraded dimethoate poorly when the chemical was used as a sole carbon and energy source, it was able to remove up to 75% of dimethoate via co-metabolism. With a response surface methodology, we optimized carbon, nitrogen and phosphorus concentrations of the media for dimethoate degradation. Raoultella sp. X1 has a potential to be a useful organism for dimethoate degradation and a model strain for studying this biological process at the molecular level.     

Chloroform degradation by butane-grown cells of Rhodococcus aetherovorans BCP1

The ability of a Rhodococcus aetherovorans strain, BCP1, to grow on butane and to degrade chloroform in the 0–633 μM range (0–75.5 mg l⁻¹) via aerobic cometabolism was investigated by means of resting-cell assays. BCP1 degraded chloroform with a complete mineralization of the organic Cl. The resulting butane and chloroform maximum specific degradation rates were equal to 118 and 22 μmol , respectively. Butane inhibition on chloroform degradation was satisfactorily interpreted by means of a model of competitive inhibition, with an inhibition constant equal to 38 % of the estimated butane half-saturation constant, whereas chloroform (at 11 μM) did not inhibit butane utilization. Acetylene (1,720 μM) induced an almost complete inactivation of the degradation of both butane and chloroform, indicating that the studied cometabolic process is mediated by a monooxygenase enzyme. BCP1 proved capable of degrading vinyl chloride and 1,1,2-trichloroethane, but not 1,2-trans-dichloroethylene. BCP1 could grow on the intermediates of the most common butane metabolic pathways and on the aliphatic hydrocarbons from ethane to n-heptane. After growth on n-hexane, it was able to deplete chloroform (13 μM) with a degradation rate higher than that obtained, at the same chloroform concentration, after growth on butane.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101

This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone–butanol–ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27–29 g l⁻¹. C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g l⁻¹. The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g l⁻¹ has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g l⁻¹ h⁻¹. Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20–0.25 lb⁻¹ by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. Journal of Industrial Microbiology & Biotechnology (2001) 27, 287–291. Received 12 September 2000/ Accepted in revised form 27 January 2001     

Biodegradation of the major color containing compounds in distillery wastewater by an aerobic bacterial culture and characterization of their metabolites

This study deals the biodegradation of the major color containing compounds extracted from distillery wastewater (DWW) by an aerobic bacterial consortium comprising Bacillus licheniformis (DQ79010), Bacillus sp. (DQ779011) and Alcaligenes sp. (DQ779012) and characterization of metabolic products. The degradation of color containing compounds by bacteria was studied by using the different carbon and nitrogen sources at different environmental conditions. Results revealed that the bacterial consortium was efficient for 70% color removal in presence of glucose (1.0%) and peptone (0.1%) at pH 7.0 and temperature 37°C. The HPLC analysis of control and bacterial degraded samples has shown the reduction in peak area as well as shifting of peaks compared to control indicating the bacterial degradation as well as transformation of color containing compounds from DWW. The comparative LC–MS–MS and other spectrophotometric analysis has shown the presence of dihydroxyconiferyl alcohol, 2, 2′-bifuran-5-carboxylic acid, 2-nitroacetophenone, p-chloroanisol, 2, 3-dimethyl-pyrazine, 2-methylhexane, methylbenzene, 2, 3-dihydro-5-methylfuran, 3-pyrroline, and acetic acid in control samples that were biodegraded and biotransformed into 2-nitroacetophenone, p-chloroanisol, 2, 2′-bifuran, indole, 2-methylhexane, and 2, 3-dihydro-5-methylfuran by bacterial consortium. In this study, it was observed that most of the compounds detected in control samples were diminished from the bacterial degraded samples and compounds 2, 2′-bifuran and indole with molecular weight 134 and 117 were produced as new metabolites during the bacterial degradation of color containing compounds from DWW.     

Antipathogenic potential of marine <Emphasis Type="Italic">Bacillus</Emphasis> sp. SS4 on <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine-lactone-mediated virulence factors production in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> (PAO1)

Antipathogenic therapy is an outcome of the quorum-sensing inhibition (QSI) mechanism, which targets autoinducer-dependent virulent gene expression in bacterial pathogens. N-acyl homoserine lactone (AHL) acts as a key regulator in the production of virulence factors and biofilm formation in Pseudomonas aeruginosa PAO1 and violacein pigment production in Chromobacterium violaceum. In the present study, the marine bacterial strain SS4 showed potential QSI activity in a concentration-dependent manner (0.5–2 mg/ml) against the AHL-mediated violacein production in C. violaceum (33–86%) and biofilm formation (33–88%), total protease (20–65%), LasA protease (59–68%), LasB elastase (36–68%), pyocyanin (17–86%) and pyoverdin productions in PAO1. The light and confocal laser scanning microscopic analyses confirmed the reduction of the biofilm-forming ability of PAO1 when treated with SS4 extract. Furthermore, the antibiofilm potential was confirmed through static biofilm ring assay, in which ethyl acetate extract of SS4 showed concentration-dependent reduction in the biofilm-forming ability of PAO1. Thus, the result of this study clearly reveals the antipathogenic and antibiofilm properties of the bacterial isolate SS4. Through 16S rDNA analysis, the strain SS4 was identified as Bacillus sp. (GenBank Accession Number: GU471751).     

Oxidative stress by biphenyl metabolites induces inhibition of bacterial cell separation

Cell–cell separation of a polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 was monitored by flow cytometry. When monohydroxy metabolites of biphenyl (BP) (2-hydroxybiphenyl and 3-hydroxybiphenyl) were added to the culture, cell–cell separation of strain TK102 was inhibited at stationary phase. This inhibition was reproduced on non-PCB degrading bacteria such as Pseudomonas putida PpY101 and Escherichia coli MV1184, but was not observed on Pseudomonas aeruginosa PAO1. An opportunistic pathogen, P. aeruginosa PAO1, produces exopolysaccharide, which is known to scavenge damaging chemicals such as reactive oxygen species (ROS). The higher level of ROS and lipid peroxidants were detected in the cells treated by monohydroxybiphenyls. Fat-soluble vitamin E, which is a lipid radical scavenger, maintained bacterial cell separation during monohydroxybiphenyls treatment. Our results demonstrated that intracellular oxidative stress played an important role in the inhibition of bacterial cell separation during BP metabolism. This study shows that metabolites of environmental pollutants, such as monohydroxylated BP, inhibit bacterial cell separation by oxidative stress.     

Preparation and chemical modification of poly-γ-l-glutamic acid

Poly-γ-l-glutamic acid was synthesized in optimized fermentor cultivation ofBacillus licheniformis. The polypeptide obtained was esterified using benzyl- and butylbromides. Benzyl esters with 30, 60 and 100% degree of esterification were prepared. Solubility of the products in dimethyl sulfoxide, chloroform, methanol and in water was significantly different from these ones of original polymer.     

Phytochemical analysis of <Emphasis Type="Italic">Andrographis paniculata</Emphasis> extract and its antimicrobial activity

The present study describes the phytochemical profile and antimicrobial activity of Andrographis paniculata. For the present investigation, two samples of A. paniculata extracts, obtained by extraction in chloroform and chloroform + HCl, respectively, were compared for their antimicrobial activity and further subjected to GC-MS analysis to find out the nature of the compounds responsible for the antimicrobial activity. The antibacterial activities were assessed by measuring the diameter of the inhibition zones, MIC and MBC values. Compared to the chloroform + HCl extract, the chloroform extract showed better antimicrobial activity against all the nine pathogenic bacterial strains tested. The chloroform extract was observed to be active against the opportunistic and pathogenic gram-negative bacteria, indicating its potential application related to noscomial infections. GC-MS results revealed phenols, aromatic carboxylic acids and esters in the chloroform extract to be the molecules responsible for the antimicrobial activity of A. paniculata. This is the first report on analysis of antimicrobial components from A. paniculata, and our results confer the utility of this plant extract in developing a novel broad spectrum antimicrobial agent.     

Pharmacodynamic parameters of aminoglycosides and their effect on exoenzymes ofPseudomonas aeruginosa

Aminoglycosides at 2× or 4× minimum inhibitory concentration induced postantibiotic effects againstPseudomonas aeruginosa lasting 3.5–4.9 h (gentamicin) and 0.5–3.7 h (selemycin). Postantibiotic effects of subinhibitory concentrations of the aminoglycosides tested were substantially longer. Some combinations of supra- and subinhibitory concentrations of antibiotics did not even allow any regrowth of the bacterial strain. The postantibiotic effects and postantibiotic effects of subinhibitory concentrations of gentamicin and selemycin were associated with changes ofP. aeruginosa elastase and proteinase. Combinations of supra- and subinhibitory concentrations more pronouncedly suppressed enzymic activities than did suprainhibitory concentrations alone.     

Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane

A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane. P. aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source. The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane). An inhibition model for P. aeruginosa grown on pentane was proposed. Pentane had an inhibitory effect on growth of P. aeruginosa, even at a concentration as low as 85 μg/l. This resulted in the calculation of the following kinetic parameters (μ_max = 0.19 h⁻¹, K _s = 2.9 μg/l, K _i = 3.5 mg/l). Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane. This model depends only on two parameters: the concentrations of pentane and MTBE. Received: 16 July 1998 / Received revision: 11 November 1998 / Accepted 31 November 1998     

Co-Utilization of Canola Oil and Glucose on the Production of a Surfactant by <Emphasis Type="Italic">Candida lipolytica</Emphasis>

Candida lipolytica synthesized a surfactant in a cultivation medium supplemented with canola oil and glucose as carbon sources. Measurements of biosurfactant production and surface tension indicated that the biosurfactant was produced at 48 h of fermentation. The surface-active species is constituted by the protein–lipid–polysaccharide complex in nature. The cell-free broth was particularly influenced by the addition of salt, the pH and temperature depending on the emulsified substrate (hexadecane or a vegetable oil). After comparison between ethyl acetate and mixtures of chloroform and methanol as solvent systems for surfactant recovery, it was found that ethyl acetate was able to extract crude surfactant material with high product recovery (8.0 g/L). The isolated biosurfactant decreased the surface tension to values of 30 mN/m at the critical micelle concentration. Emulsification properties of the biosurfactant produced were compared to those of commercial emulsifiers and other microbial surfactants.