Determination of minimal concentrations of biocorrosion inhibitors by a bioluminescence method in relation to bacteria, participating in biocorrosion

By using a bioluminescence ATP assay, we have determined the minimal concentrations of some biocorrosion inhibitors (Katon, Khazar, VFIKS-82, Nitro-1, Kaspii-2, and Kaspii-4) suppressing most common microbial corrosion agents: Desulfovibrio desulfuricans, Desulfovibrio vulgaris, Pseudomonas putida, Pseudomonas fluorescens, and Acidithiobacillus ferrooxidans. The cell titers determined by the bioluminescence method, including not only dividing cells but also their dormant living counterparts, are two- to sixfold greater than the values determined microbiologically. It is shown that the bioluminescence method can be applied to determination of cell titers in samples of oil-field waters in the presence of iron ions (up to 260 mM) and iron sulfide (to 186 mg/l) and in the absence or presence of biocidal corrosion inhibitors.     

The Development of a Bioluminescence Assay to Compare the Efficacy of Biocides Incorporated into Plasticised PVC

A bioluminescence assay was developed using the expression of the luxAB genes in Pseudomonas veronii to allow the efficacy of biocides incorporated into plasticised polyvinylchloride (pPVC) to be determined in situ. A maximum number of cells was found to adhere to the surface after 18 h as measured by bioluminescence, radiolabelling and viable cell counts. A positive correlation was found between the level of bioluminescence and numbers of viable cells attached to the pPVC. When the biocide 10, 10-oxybisphenoxyarsine (OBPA) was incorporated into the pPVC, both bioluminescence and viable cell number were reduced by ca 60% at a concentration of 750 ppm and by >99% at 2250 ppm. When the biocide 2,3,5,6-tetrachloro-4-(methylsulphonyl)pyridine (TCMP) was incorporated into the pPVC, no reduction in viability or bioluminescence was seen after 18 h. However, over a period of 72 h at a concentration of 2250 ppm TCMP, both viable cell number and bioluminescence decreased steadily after 36 h until after 72 h, both bioluminescence and viable cell counts were less than 1% of the initial values. The viability of attached cells can therefore be measured in situ in a sensitive real-time assay by measuring bioluminescence allowing the efficacy of biocides incorporated into plastics to be compared.     

Bioluminescence of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> HK44 in the course of encapsulation into silica gel. Effect of methanol

The bioluminescence (BLM) and colony-forming units (CFU) of Pseudomonas fluorescens HK44 were monitored during encapsulation into pre-polymerized Si(OMe)₄. The non-induced BLM of free cells was increased in the presence of 0.5–2.5 % MeOH. After mixing silica sol with the cell suspension, both BLM and CFU dropped to 1–3 and 8–18 %, respectively; both remained lowered as long as the silica biofilm contained residual MeOH. The kinetics of MeOH being released from silica biofilms (a thickness of 2–6 mm) were first-order. The decrease of bacterial activity due to encapsulation was proportional to the biofilm thickness. MeOH evolving during encapsulation is probably the principal stress factor but not the only one.     

Influence of inoculum density of the antagonistic bacteria Pseudomonas fluorescens and Pseudomonas corrugata on sugar beet seedling colonisation and suppression of Pythium damping off

The effect of initial inoculum density of the antagonistic bacterial strains Pseudomonas fluorescens B5 and Pseudomonas corrugata 2140 (10³ to 10⁸ CFU per seed pellet) on sugar beet seedling colonisation, in situ bioluminescence and antagonistic activity towards Pythium ultimum was investigated. Populations of the bacteria colonising sugar beet root systems approached an apparent carrying capacity of 10⁵ to 10⁶ CFU per plant after 12 d growth, irrespective of inoculum density. This meant an up to 320-fold population increase at low inoculum densities and a decrease at high densities. Population densities of both bacteria and their corresponding in situ bioluminescence (resulting from luciferase enzyme expression from the inserted luxAB genes) reached highest levels in the hypocotyl region and in the upper root region 0–20 mm below seed level (10⁴–10⁶ CFU/cm section, 10¹–10³ RLU/cm section) and decreased with root depth. In situ bioluminescence, which indicates physiological activity, was measurable at lowest antagonist initial inoculum density (10³ CFU per seed pellet) and did not increase significantly with increasing inoculum density. Bioluminescence was also significantly correlated with population density. For Pseudomonas fluorescens B5, the total population size per plant and downward colonisation of the root (below 40 mm depth) increased significantly with antagonist inoculum density applied to the seeds. For Pseudomonas corrugata 2140, no significant influence of initial inoculum density on root colonisation was observable. Survival and dry weight of sugar beet seedlings in Pythium infested soil increased significantly with increasing inoculum density of Pseudomonas fluorescens B5, whereas for Pseudomonas corrugata 2140, initial densities of 10⁴ to 10⁶ CFU per seed resulted in maximal survival of plants.     

Axenic aerobic biofilms inhibit corrosion of copper and aluminum

The corrosion behavior of unalloyed copper and aluminum alloy 2024 in modified Baar's medium has been studied with continuous reactors using electrochemical impedance spectroscopy. An axenic aerobic biofilm of either Pseudomonas fragi K or Bacillus brevis 18 was able to lessen corrosion as evidenced by a consistent 20-fold increase in the low-frequency impedance value of copper as well as by a consistent four- to seven-fold increase in the polarization resistance of aluminum 2024 after six days exposure compared to sterile controls. This is the first report of axenic aerobic biofilms inhibiting generalized corrosion of copper and aluminum. Addition of the representative sulfate-reducing bacterium (SRB) Desulfovibrio vulgaris (to simulate consortia corrosion behavior) to either the P. fragi K or B. brevis 18 protective biofilm on copper increased the corrosion to that of the sterile control unless antibiotic (ampicillin) was added to inhibit the growth of SRB in the biofilm. Received: 24 May 1999 / Received revision: 6 July 1999 / Accepted: 1 August 1999     

Sulphate-reducing bacteria which do not induce accelerated corrosion

The corrosive activity of the recently isolated SRB genera has not previously been reported in the literature. In this investigation, three genera of sulphate-reducing bacteria (SRB). Desulfovibrio vulgaris, Desulfobacter postgatei and Desulfobulbus propionicus were tested for their ability to induce accelerated corrosion of mild steel in laboratory growth media. Desulfovibrio vulgaris, well-recognised for its corrosive activity, caused a 78·2% increase in weight loss compared to the control, uninoculated medium (95% confidence limits +37·0% to +130·6%). Desulfobacter postgatei and Desulfobulbus propionicus had no significant effect on corrosion. For Desulfobacter postgatei the mean rate of corrosion was 10·6% more than the control (95% confidence limits −12·0% to +39·0%). For Desulfobulbus propionicus the mean corrosion rate was increased by 5·8% over control (95% confidence limits −9·8% to +24·2%).     

Chelators enhanced biocide inhibition of planktonic sulfate-reducing bacterial growth

Biocides are currently the primary mitigation method to control sulfate-reducing bacteria (SRB) in biofouling, reservoir souring and microbiologically influenced corrosion. Increasingly restrictive environmental regulations and safety concerns on biocide uses demand more efficient dosing of biocides. Chelators have been known to enhance antibiotics because of their properties such as increasing the permeability of the outer cell membrane of Gram-negative bacteria. Two readily biodegradable chelators, ethylenediaminedisuccinate (EDDS) and N-(2-hydroxyethyl)iminodiacetic acid (HEIDA) disodium salts that are touted as potential replacements of ethylenediaminetetraacetic acid (EDTA), were evaluated as potential biocide enhancers for glutaraldehyde and tetrakis hydroxymethyl phosphonium sulfate (THPS) in their inhibition of planktonic SRB growth. Desulfovibrio vulgaris ATCC 7757 and Desulfovibrio desulfuricans ATCC 14563 were grown in modified ATCC 1249 medium and in enriched artificial seawater, respectively. Laboratory tests in 100 ml anaerobic vials showed that EDDS or HEIDA alone did not inhibit SRB growth. However, when EDDS or HEIDA was combined with glutaraldehyde or THPS, each of them enhanced the biocide inhibition of planktonic SRB growth.     

Direct cathodic electron uptake coupled to sulfate reduction by Desulfovibrio ferrophilus IS5 biofilms

Direct electron uptake is emerging as a key process for electron transfer in anaerobic microbial communities, both between species and from extracellular sources, such as zero-valent iron (Fe⁰) or cathodic surfaces. In this study, we investigated cathodic electron uptake by Fe⁰-corroding Desulfovibrio ferrophilus IS5 and showed that electron uptake is dependent on direct cell contact via a biofilm on the cathode surface rather than through secreted intermediates. Induction of cathodic electron uptake by lactate-starved D. ferrophilus IS5 cells resulted in the expression of all components necessary for electron uptake; however, protein synthesis was required for full biofilm formation. Notably, proteinase K treatment uncoupled electron uptake from biofilm formation, likely through proteolytic degradation of proteinaceous components of the electron uptake machinery. We also showed that cathodic electron uptake is dependent on SO₄²⁻ reduction. The insensitivity of Fe⁰ corrosion to proteinase K treatment suggests that electron uptake from a cathode might involve different mechanism(s) than those involved in Fe⁰ corrosion.     

A Pseudomonas aeruginosa biosensor responds to exposure to ultraviolet radiation

We fused the Pseudomonas aeruginosa recA promoter to a promoterless Vibrio fisherilux operon. This recA–lux fusion (pMOE15) was introduced into wild-type P. aeruginosa strain FRD1 and recA expression was monitored by measuring 490-nm light production. The RM4440 strain responded to increasing doses of ultraviolet radiation by an increase in its bioluminescence. RM4440 has the potential to be useful as a biosensor for the presence of DNA-damaging agents in the environment. Received: 18 February 1998 / Received revision: 18 June 1998 / Accepted: 27 June 1998     

Inhibiting sulfate-reducing bacteria in biofilms on steel with antimicrobial peptides generated in situ

In batch and continuous fermentations, the reduction in corrosion of SAE 1018 mild steel and 304 stainless steel caused by inhibition of the reference sulfate-reducing bacterium (SRB) Desulfovibrio vulgaris by a protective, antimicrobial-producing Bacillus brevis biofilm was investigated. The presence of D. vulgaris produced a thick black precipitate on mild steel and a higher corrosion rate in batch cultures than that seen in a mono-culture of non-antimicrobial-producing Pseudomonas fragi K upon the addition of SRB to the aerobic P. fragi K biofilm. In continuous reactors, the polarization resistance R _p decreased for stainless steel and increased for mild steel upon the addition of SRB to a P. fragi K biofilm. Addition of either 200 μg/ml ampicillin, chloramphenicol, or ammonium molybdate to batch and continuous reactors after SRB had colonized the metal was ineffective in killing SRB, as inferred from the lack of change in both R _p and the impedance spectra. However, when ampicillin was added prior to SRB colonization, the growth of SRB was completely inhibited on stainless steel in continuous reactors. Prior addition of ampicillin was only able to delay the growth of SRB on mild steel in continuous reactors. External addition of the purified peptide antimicrobial agent gramicidin S prior to the addition of SRB also inhibited the growth of SRB on stainless steel in continuous reactors, and the SRB were also inhibited on stainless steel in both batch and continuous reactors by producing gramicidin S in situ in a protective biofilm when the gramicidin-S-overproducing strain Bacillus brevis 18 was used. Received: 29 October 1998 / Received revision: 18 February 1999 / Accepted: 26 February 1999     

Regulation of the Periplasmic [Fe] Hydrogenase by Ferrous Iron in Desulfovibrio vulgaris (Hildenborough)

The periplasmic [Fe] hydrogenase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) DSM 8303 was found to be regulated by ferrous iron availability. During growth with 5 ppm of iron, the enzyme derepressed and the specific activity increased approximately fourfold, whereas the presence of 100 ppm of ferrous iron repressed the enzyme. The repression-derepression phenomenon with ferrous iron was found to be operative when the cells were cultured under either hydrogen or nitrogen gas. This is the first reported case showing that the hydrogenase enzyme is regulated by iron, and the implications of this finding relative to the corrosion industry are discussed.     

A combination of NADHP and hispidin is not essential for bioluminescence in luminous fungal living gills of Mycena chlorophos

The chemical mechanisms underlying visible bioluminescence in the fungus Mycena chlorophos are not clear. A combination of dihydronicotinamide adenine dinucleotide phosphate (NADPH) and hispidin, which has been reported to increase the intensity of in vitro luminescence in crude cold‐water extracts prepared from the bioluminescent fruiting bodies of M. chlorophos, exhibited potential bioluminescence activation in the early bioluminescence stages, in which the bioluminescence was ultra‐weak, for living gills and luminescence activation for non‐bioluminescent gills, which was collapsed by freezing and subsequent thawing, at all bioluminescence stages. These abilities were not evident in considerably bioluminescent gills. These abilities were blocked by trans‐4‐hydroxycinnamic acid and trans‐3,4‐dihydroxycinnamic acid, which were identified as in vivo bioluminescence‐activating components. Original bioluminescence and bioluminescence produced from the addition of trans‐4‐hydroxycinnamic acid and trans‐3,4‐dihydroxycinnamic acid in living gills were almost completely inhibited by 10 mM NaN₃, whereas the luminescence produced form the combination of NADPH and hispidin in thawed non‐bioluminescent and living gills at the early weak bioluminescence stages was not inhibited by 10 mM NaN₃. Thus, the combination of NADPH and hispidin plays different roles in luminescence systems compared with essential bioluminescence systems, and the combination of NADPH and hispidin was not essential for visible bioluminescence in living gills.     

Determination of non-protein-bound iron in human synovial fluid by high-performance liquid chromatography with electrochemical detection

Non-protein-bound iron in human synovial fluid was determined using high-performance liquid chromatography with electrochemical detection. The procedure was based on the separation of the iron—diethylenetriaminepentaacetic acid (DPTA) complex formed directly on a chromatographic column containing an anion-exchange resin followed by electrochemical detection. The method enabled more than 0.1 μM Fe(III) to be determined with an injection volume of 10 μl. A mixture of synovial fluid, 20 μM DTPA and acetate buffer was incubated in the presence and absence of superoxide (O⁻₂) generated by a xanthine—xanthine oxidase system and was ultrafiltered through a 30 000 molecular mass cut-off filter. No iron was detected in the ultrafiltrate at physiological pH. However, the presence of iron was observed in the ultrafiltrate at low pH, and O⁻₂ and decreased pH, iron may be released into the synovial fluid.     

Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellate Lingulodinium polyedrum: evidence for the role of stretch‐activated ion channels

Dinoflagellate bioluminescence serves as a whole‐cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage‐sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200 μM Gadolinium III (Gd³⁺), a nonspecific blocker of stretch‐activated and some voltage‐gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50 μM nifedipine, an inhibitor of L‐type voltage‐gated Ca²⁺ channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1 mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol‐stimulated bioluminescence was inhibited by Gd³⁺ but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch‐activated and voltage‐gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch‐activated component at the plasma membrane.     

Establishment of an EC50 database of pesticides using a Vibrio fischeri bioluminescence method

An EC₅₀ database was established to assess the acute toxicity of 16 PESTANAL pesticide standards and of seven pesticide commercial formulations using a Vibrio fischeri bioluminescence method. Half maximal effective concentration ( EC₅₀) is defined as the concentration of pollutant (in this case, pesticide) destroying 50% of the bacteria population and causing 50% bioluminescence inhibition, after a specified exposure time. Linear curves of bioluminescence inhibition versus pesticide concentration and EC₅₀ values were obtained for exposure times (t) of 5 or 15 min for these pesticides. The EC₅₀ values ranged from 6.90 × 10^?4 to 0.83 mg/ml (t = 5 min), and from 9.00 × 10^?4 to 0.37 mg/ml (t = 15 min) for pesticide standards, plus from 0.0077 to 0.74 mg/ml (t = 5 min), and from 0.0076 and 0.57 mg/ml (t = 15 min) for pesticide commercial formulations. The EC₅₀ database allowed classification of the pesticides under study into three categories according to their toxicity: very toxic, toxic and moderately toxic. These results demonstrated that the establishment of an EC₅₀ database and of linear curves of bioluminescence inhibition versus the pesticide concentration resulted in very important and irreplaceable tools to estimate the global and individual toxicity of pesticides present in environmental samples.     

New Analysis Method for Protein Phosphatase Type 2A Inhibitors Using the Firefly Bioluminescence System

A new analysis method for protein phosphatase type 2A inhibitors was established that uses the firefly bioluminescence system for detection. Thus, firefly luciferin phosphate was used as a substrate, and the liberated free luciferin was determined from the amount of light emitted from the immobilized luciferase. This method was successfully used to determine the activities of known inhibitors, i.e., okadaic acid, calyculin A, microcystin-LR and tautomycin using less than 10 pmol of a sample.     

Sensitive Analysis of Protein Phosphatase Inhibitors by the Firefly Bioluminescence System: Application to PP1γ

Protein phosphatases are classified into types 1 (PP1) and 2 (PP2A, 2B, and 2C). We have already established a new analysis method for PP2A inhibitors by using the firefly bioluminescence system for detection. This method was successfully applied to determine the PP1γ inhibitory activity of known inhibitors, i.e., calyculin A, microcystin-LR, and tautomycin, requiring less than 10 pmol of a sample.