Impact of electrokinetic remediation on microbial communities within PCP contaminated soil.

Electrokinetic techniques have been used to stimulate the removal of organic pollutants within soil, by directing contaminant migration to where remediation may be more easily achieved. The effect of this and other physical remediation techniques on the health of soil microbial communities has been poorly studied and indeed, largely ignored. This study reports the impact on soil microbial communities during the application of an electric field within ex situ laboratory soil microcosms contaminated with pentachlorophenol (PCP; 100mg kg(-1) oven dry soil). Electrokinetics reduced counts of culturable bacteria and fungi, soil microbial respiration and carbon substrate utilisation, especially close to the acidic anode where PCP accumulated (36d), perhaps exacerbated by the greater toxicity of PCP at lower soil pH. There is little doubt that a better awareness of the interactions between soil electrokinetic processes and microbial communities is key to improving the efficacy and sustainability of this remediation strategy.

Selection of microbial consortia for treating metal-working fluids.

The aim of this research was to determine the effectiveness of a strategy for constructing microbial consortia for treating chemically mixed industrial effluent, based on a more thorough understanding of communities within waste metal-working fluids (MWFs). Complementary phenotypic and genotypic methods revealed that the microbial communities in spent MWFs had low diversity and were very similar in species composition in samples originating from different locations and uses. Of 65 bacterial isolates studied, only 9 species were identified using fatty acid methyl ester (FAME) analysis. The results of genotypic analysis by denaturing gradient gel electrophoresis (DGGE) were congruent with observations made using FAME analysis. The metabolic potential of the isolates was assessed in terms of assimilation ability and tolerance of co-contaminants. The three isolates, selected (Clavibacter michiganensis, Methylobacterium mesophilicum, and Rhodococcus erythropolis) to form a consortium, were representative of three of the four most abundant populations and when combined could utilise or tolerate all of the individual MWF components, including the biocide and the recalcitrant compound benzotriazole.

Substrate-regulated cyanide hydratase (chy) gene expression in Fusarium solani: the potential of a transcription-based assay for monitoring the biotransformation of cyanide complexes.

The fungus Fusarium solani detoxifies cyanide through induction of the cyanide hydratase gene activity (chy) in the presence of either KCN or the metal-complexed cyanides, K2Ni(CN)4 or K4Fe(CN)6, at pH 7.0 and 4.0 respectively. Sequence analysis of the chy gene identified primers for reverse transcriptase-polymerase chain reaction (RT-PCR)-directed analysis of mRNA transcripts, which demonstrated that activity correlated to the substrate-specific induction of gene expression. chy transcription was initiated 30-60 min after exposure of F. solani cultures to cyanide complexes. Maximum expression was detected within 4.5 h, after which chy mRNA synthesis declined below the limits of detection within 26 h. A lag period of approximately 2 h, following initial transcription, was recorded before cyanide complexes were converted to formamide. mRNA transcripts of chy were not detected in the absence of cyanide or cyanide complexes. The presence of introns within the gene resulted in a difference in size of 100 bp for DNA compared with mRNA of the corresponding 5' region. This size difference facilitated PCR detection of gene and transcript respectively. Comparisons of the predicted amino acid sequence of the F. solani chy gene and those of Gloeocerospora sorghi, Fusarium lateritium and Leptosphaeria maculans demonstrate that cyanide hydratase genes are highly conserved and of a similar evolutionary origin. These data predict that the functional assay described here to monitor the induction of chy gene expression and, potentially, cyanide degradation would be applicable to a variety of polluted environments.

Regioselective biotransformation of the dinitrile compounds 2-, 3- and 4-(cyanomethyl) benzonitrile by the soil bacterium Rhodococcus rhodochrous LL100-21.

The cyanomethyl benzonitrile compounds used for this study contain two cyano groups: a -CH(2)CN side chain, plus a cyano group attached to the benzene ring. The ortho, meta and para -CH(2)CN substituted compounds were biotransformed using whole cell suspensions of the bacterium Rhodococcus rhodochrous LL100-21. The bacterium had previously been grown on the mono-nitrile compounds propionitrile, benzonitrile or acetonitrile, inducing the formation of nitrile hydrolyzing enzymes.Suspensions of R. rhodochrous LL100-21 that had been grown on propionitrile or benzonitrile converted the aliphatic group of 2-(cyanomethyl) benzonitrile (a) to the corresponding carboxylic acid, 2-(cyanophenyl) acetic acid (d) with excellent recovery of the product and no evidence for any other products. Conversely, when grown on acetonitrile the bacterium converted 2-(cyanomethyl) benzonitrile (a) to the amide derivatives 2-(cyanophenyl) acetamide (k) and 2-(cyanomethyl) benzamide (l) but only in low yields.Biotransformations of 3-(cyanomethyl) benzonitrile (b) and 4-(cyanomethyl) benzonitrile (c), by suspensions of bacteria that had been grown on benzonitrile or propionitrile, resulted in hydrolysis of the aromatic nitrile to produce 3- and 4-(cyanomethyl) benzoic acid (j) and (m), respectively, both with a high yield. Low concentrations of other products were also detected, for example the diacids 3- and 4-(carboxyphenyl) acetic acid (h) and (i).When the bacterium was grown on acetonitrile it could biotransform 3- and 4-(cyanomethyl) benzonitrile (b) and (c) to different products indicating less regiospecificity by the nitrile hydratase enzyme.Comparison of the initial rates of conversion of the aliphatic cyano side chain of 2-(cyanomethyl) benzonitrile (a) and other substituted benzonitriles indicated that electronic effects did not affect the initial rate of the reaction as they would require transmission through an SP(3) methylene carbon atom.

Electrokinetic movement and biodegradation of 2,4-dichlorophenoxyacetic acid in silt soil.

The coupling of electrokinetic movement of an organic contaminant, 2,4-dichlorophenoxyacetic acid (2,4-D), through soil and its biodegradation in situ has been demonstrated. In a first experiment, the direction and rate of movement of 2,4-D were determined using homogeneously contaminated soil (864 mg 2,4-D/kg dry weight soil) compacted into six individual compartments, 6 cm long, 3 cm wide, and 4 cm deep. Each compartment was bordered by a carbon felt anode and a stainless steel cathode. The application of a current density of 3.72 A/m(2) led to migration of 2,4-D towards the anode at a rate of approximately 4 cm/day. In a second experiment, electrokinetic movement and biodegradation were combined in situ. Sterilized silt soil contaminated with ring-labeled 14C-2,4-D (811 mg 2,4-D/kg dry weight soil) was compacted into a single soil compartment, 22 cm long, 7 cm wide, and 4 cm deep, in a 4.5 cm region adjacent to the cathode. The remainder of the compartment was filled with sterilized soil (to a total weight of 1,015 g). Burkholderia spp. RASC c2 (1.88 x 10(11) cells), a tetracycline-resistant bacterium with chromosomally encoded degradative genes for 2,4-D, was inoculated into the soil at a position 14-16 cm from the cathode. The reactor was placed within a sealed perspex box, with a constant air flow connected to sodium hydroxide traps. Under an applied current density of 0.89 A/m(2), the pollutant moved towards the bacteria. As it reached the inoculated region, its concentration decreased in the soil and 14CO2 was recovered in the traps. At the end of the experiment, 87.1% of radiolabel had been removed from the soil, 5.8% of which was recovered as 14CO2. A third, control, experiment showed a significant contrast in the absence of an electric current, where a slow rate of diffusion controlled the movement of both 2,4-D and bacteria in the soil and biodegradation occurred at the interface between the diffusing fronts.

Utilisation of aliphatic compounds by acidophilic heterotrophic bacteria. The potential for bioremediation of acidic wastewaters contaminated with toxic organic compounds and heavy metals.

Acidophilic, heterotrophic bacteria isolated from acidic mine effluent metabolised a range of aliphatic compounds. Aliphatic acids, which are normally thought to be toxic to acidophiles, were utilised as substrates for energy and growth by these bacteria. This biodegradative ability, concomitant with their tolerance of heavy metals, has demonstrated the potential for using these organisms for the bioremediation of multiply contaminated acidic wastewaters.

Effect of metal complexation on the bioavailability of nitrilotriacetic acid to Chelatobacter heintzii ATCC 29600.

Many polluted sites contain a mixture of organics and heavy metals. Nitrilotriacetic acid has been chosen as a model organic compound to study the effect of metal binding on organic bioavailability and degradation of organics. The effect of varying the ratio of metal to nitrilotriacetic acid on its utilisation has been examined using the gram-negative bacterium Chelatobacter heintzii ATCC 29600. The following parameters of substrate utilisation were examined: growth, degradation, respiration, mineralisation and nitrilotriacetic acid uptake. Complexation of nitrilotriacetic acid by Cu(II), Ni(II), Co(II) and Zn(II) prevented utilisation of nitrilotriacetic acid by C. heintzii; complexation to Fe(III) or Mn(II) did not. The pattern of inhibition was consistent with a 1:1 stoichiometry of metal binding to nitrilotriacetic acid. Inhibition was not due to metal ion toxicity, but was a result of metal-nitrilotriacetic acid complexes being recalcitrant to degradation. In addition, the effect of complexing (phosphate) and non-complexing (PIPES) buffers on bioavailability was examined: Co and Zn prevented degradation of nitrilotriacetic acid in PIPES buffer, but not in phosphate buffer. This was due to the removal of Co and Zn from solution by phosphate precipitation, leaving nitrilotriacetic acid uncomplexed. The results demonstrated that metal-organic complexation can alter the bioavailability of organic pollutants and may also modulate the toxicity of heavy metals.

Real-time monitoring of nitrile biotransformations by mid-infrared spectroscopy.

In this study mid-infrared spectroscopy was used to follow the enzyme kinetics involved in nitrile biocatalysis using whole cell suspensions of the bacterium Rhodococcus rhodochrous LL100-21. The bacteria were grown on acetonitrile to induce a two-step enzymatic pathway. Acetonitrile was biotransformed to acetamide by a nitrile hydratase enzyme and subsequently to acetic acid (carboxylate ion) by an amidase enzyme. The bacteria were also grown on benzonitrile to induce a one-step enzymatic pathway. Benzonitrile was biotransformed directly to benzoic acid (carboxylate ion) by a nitrilase enzyme. These reactions were followed by React IR using a silicon probe and gave excellent quantitative and qualitative real-time data of both nitrile biocatalytic reactions. This study has shown that this novel technique has potentially useful applications in biocatalysis.

The biotransformation of t-butylacetonitrile and its boron-containing analogue trimethylamine-cyanoborane by Brevibacterium R312.

The ability of the nitrile hydratase/amidase system from Brevibacterium R312 to biotransform tert-butylacetonitrile was studied with a view to their utilisation in the production of novel amino acids from isostructural compounds. Brevibacterium R312 was able to transform nitriles with this structure; however, the wide spectrum amidase from this organism was unable to biotransform the corresponding amide to the carboxylic acid.

Influence of chlorobenzoates on the utilisation of chlorobiphenyls and chlorobenzoate mixtures by chlorobiphenyl/chlorobenzoate-mineralising hybrid bacterial strains.

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 microM) and 3,4-CBA (50 microM). Although 2,5-cba and 2,6-cba were not utilised as carbon sources by strain jhr22, they did not inhibit 2-cba utilisation at the concentrations studied, whereas 2,4-cba was co-metabolised with 2-cba. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.

The development of a novel strategy for the microbial treatment of acrylonitrile effluents.

Effluent from the manufacture of acrylonitrile is difficult to biodegrade. It contains nine major organic components: acetic acid, acrylonitrile, acrylamide, acrylic acid, acrolein, cyanopyridine, fumaronitrile, succinonitrile, and maleimide. A range of bacteria have been isolated that can grow on, or convert all of the organic components of effluent from the manufacture of acrylonitrile. These bacteria can be used as the basis of a mixed culture system to treat the effluent. The bacteria were utilised in batch and continuous cultures to degrade a synthetic wastewater containing acrylonitrile, acrylamide, acrylic acid, cyanopyridine and succinonitrile. The mixed microbial population was adapted by varying the growth rate and switching from continuous to batch and back to continuous growth, to degrade these five compounds as well as acrolein, fumaronitrile and maleimide.

The utilization of thiocyanate as a nitrogen source by a heterotrophic bacterium: the degradative pathway involves formation of ammonia and tetrathionate.

A Gram-negative soil bacterium (isolate 26B) has been shown to utilize up to 100 mM thiocyanate as a source of nitrogen when supplied with glucose as the source of carbon and energy. During growth of isolate 26B with thiocyanate as the source of nitrogen, no ammonia, nitrate, nitrite, cyanide, cyanate, sulfate, sulfite, sulfide or carbonyl sulfide was detected in the growth medium. Growth of the bacterium on 14C-labelled thiocyanate (1.6 microCi) and glucose, yielded 14C-labelled carbon dioxide (0.9 microCi). The addition of 2.9 mM thiocyanate to a bacterial suspension in phosphate buffer (50 mM, pH 7.4) resulted in the utilization of 2.1 mM thiocyanate and the production of 2.0 mM ammonia. This activity was inducible and only occurred after growth of the bacterium with thiocyanate as the source of nitrogen. Tetrathionate (0.7 mM) was detected in the medium after the utilization of thiocyanate (2.4 mM) by a suspension of the bacterium in phosphate buffer, and thiosulfate (1.0 mM) was detected as an intermediate. The addition of sulfide or thiosulfate to the bacterial suspension also resulted in the formation of tetrathionate. The utilization of both of these compounds appeared to be constitutive. A pathway for thiocyanate utilization by isolate 26B is proposed which involves the hydrolysis of thiocyanate to produce cyanate and sulfide. The cyanate then undergoes further hydrolysis to form ammonia and carbon dioxide. The sulfide is ultimately oxidized to tetrathionate via a pathway which includes thiosulfate.

Mammalian cell damage in a novel membrane bioreactor.

The experimental study has assessed a novel membrane bioreactor for mammalian cell culture. In the absence of a gas phase, the key features of cell damage associated with laminar and turbulent flow have been identified. The bioreactor employs a dimpled membrane in order to enhance transverse mixing in a narrow channel, but a fall in viable cell density has been observed at Reynolds numbers above Re = 83. In the laminar flow regime wall shear is the critical mechanism and an accurate calculation of shear rate in a complex channel has been achieved using the Reynolds analogy. Flow generating a wall shear rate in excess of 3000 s(-1) has been shown to cause damage. Power dissipation measurements have been used to distinguish between laminar and turbulent flow and also to predict Kolmogorov eddy lengths. An additional turbulent bulk stress damage mechanism at higher Reynolds numbers (Re > 250) results in a very rapid fall in viable cell density.

A novel membrane bioreactor for microbial growth.

A novel membrane bioreactor, previously assessed for its gas transfer characteristics, was used in various size and membrane configurations for the growth of the strictly aerobic bacterium Pseudomonas aeruginosa. The bioreactor was found to readily support growth, and the initial growth rates showed the previously demonstrated enhanced effect in gas O2 mass transfer of the dimpled membrane bioreactor over flat membrane bioreactors. The production of a secondary metabolite by a Pseudomonas sp. following growth was demonstrated, as was the biotransformation of a nitrile by Nocardia rhodochrous with the removal of the biotransformation products across a membrane. The potential of the bioreactor, in terms of other applications in the field of biotechnology, is discussed.

Gas transfer characteristics of a novel membrane bioreactor.

We show the design features of a membrane bioreactor based on pulsatile flow across dimpled membranes. Results show an enhanced mass transfer of air of at least five-fold magnitude as compared with flat membranes. An increased working volume form 20 mL to 120 mL reduced the k(L)A at a given Reynolds number because of axial mixing of fluid from the deoxygenated end chamber. The bioreactor was used to supply air to a hybridoma mammalian cell line, and the calculated oxygen uptake showed that high-density cultures could be maintained in a 20 mL, single-dimpled cultures could be maintained in a 20 mL, single-dimpled membrane system. Indirect aeration of a 2 L continuous stirred tank reactor, by a double-membrane system, showed that air could be supplied to mammalian cells at cell densities of approximately 4 x 10(6) /mL.

The anaerobic utilisation of cyanide in the presence of sugars by microbial cultures can involve an abiotic process.

The degradation of cyanide under anaerobic conditions in the presence of a growing culture of a strain of Klebsiella planticola has been shown to be due to a chemical process dependent upon the presence of a reducing sugar in the medium. The conversion of cyanide to ammonia was independent of any biological factors under these conditions.

Effects of auricular acupuncture-like transcutaneous electric nerve stimulation on pain levels following wound care in patients with burns: a pilot study.

This study was designed to test the hypothesis that auricular acupuncture-like transcutaneous electrical nerve stimulation would significantly reduce the pain experienced by patients with burns immediately after wound debridement, other wound care, and dressing changes. Subjects were 11 inpatients at the University of Alabama Hospital Burn Unit. A two-period crossover design was used, and each patient received one experimental treatment consisting of bilateral acupuncture-like transcutaneous electrical nerve stimulation to six ear points and one control treatment consisting of a placebo pill. The Visual Analogue Scale was used as a measure of pain and was completed immediately before and after treatments and at 15, 30, and 60 minutes after treatment. A two-factor repeated measures ANOVA indicated significant effects of measurement time (p less than 0.001) and treatment by time (p = 0.002). Post hoc analysis revealed significant differences (p less than 0.05) between experimental and control conditions at all times after treatment but not at pretreatment baseline. These results indicate that auricular acupuncture-like transcutaneous electrical nerve stimulation may be an effective pain management technique in patients with burns.

Relationship of professional involvement to clinical behaviors of physical therapists.

This study was designed to measure the level of professional involvement of respondents and to examine the relationship between a high level of professional involvement (eg, membership in the American Physical Therapy Association, attendance at Association conferences, participation in continuing education programs) and the frequency of use of high-complexity procedures. A questionnaire survey of 165 Alabama physical therapists was conducted to establish 1) the ranking of 24 physical therapy procedures according to the perceived level of complexity and 2) the relationship of a procedure's rank to delegation of that procedure to support personnel. The ranking and a cluster analysis of the 24 procedures correlated with similar ranking in other studies (Pearson r = .92). The level of complexity of a procedure influenced delegation to support personnel, with high-complexity procedures being delegated less frequently and requiring greater supervision than moderate- and low-complexity procedures. There was variation in the frequency of use of procedures, but a clear relationship was not found between a high level of professional involvement and use of high-complexity procedures. A relationship existed between professional involvement and both job title and types of referrals received. Those physical therapists with a higher level of professional involvement tended to be directors and supervisors (chi 2 = 28.29, df = 2, p less than .001), and they received autonomous referrals with greater frequency than those physical therapists with a lower level of involvement (chi 2 = 16.25, df = 3, p less than .001). The findings have implications for the continued professionalization of physical therapy and for the role that physical therapy educators and administrators play in this process.

Effect of helium-neon laser auriculotherapy on experimental pain threshold.

This study was conducted to examine the effects of helium-neon laser auriculotherapy on experimental pain threshold. Eighty healthy female and male subjects, aged 18 to 39 years, were assigned randomly to one of two treatment groups. Subjects in the Experimental Group (n = 41) received laser stimulation, and subjects in the Control Group (n = 39) received sham stimulation to appropriate acupuncture points on the left ear. Experimental pain threshold at the ipsilateral wrist was determined with an electrical stimulus immediately before and after treatment. The mean change (posttreatment minus pretreatment) for the Experimental Group was greater than the mean change for the Control Group (p less than .05). The Experimental Group demonstrated a statistically significant (p less than .05) increase in mean pain threshold after treatment, but the Control Group did not. Results indicate that helium-neon laser auriculotherapy can increase experimental pain threshold and suggest a possible alternative for patients intolerant of transcutaneous electrical nerve stimulation.

Comparison of effects of transcutaneous electrical nerve stimulation of auricular, somatic, and the combination of auricular and somatic acupuncture points on experimental pain threshold.

This study compared the effects of high intensity, low frequency transcutaneous electrical nerve stimulation of auricular, somatic, and combined auricular and somatic acupuncture points on experimental pain threshold measured at the wrist. Sixty-seven healthy adults, aged 18 to 39 years, were assigned randomly to one of four groups: 1) the Auricular Group (n = 17) received TENS to auricular acupuncture points, 2) the Somatic Group (n = 17) received TENS to somatic acupuncture points, 3) the Combined Group (n = 17) received TENS to both auricular and somatic acupuncture points, and 4) the Control Group (n = 16) received no TENS and served as controls. Pain threshold was measured immediately before and after treatment or rest. Pain threshold significantly increased (p less than .05) in the Auricular, Somatic, and Combined Groups following treatment, with no statistically significant differences in mean pain threshold change scores among treatment groups. The Control Group demonstrated no statistically significant change in pain threshold. The results indicate that TENS applied to any of the three sets of acupuncture points equally increases pain threshold, thus possibly increasing options in choosing stimulation sites for treating patients with pain.