Christopher J. Knowles

Biodegradation of metal cyanides by mixed and pure cultures of fungi

Abstract Former gasworks sites are sometimes be heavily contaminated with spent oxide which contains cyanide complexed to metals (especially iron). In this study, mixed fungal cultures have been isolated from acidic gasworks soil by their ability to utilize iron or nickel cyanide as the sole source of nitrogen at acidic or neutral pH, respectively. A mixed culture comprising Fusarium solani and Trichoderma polysporum was obtained by enrichment on tetracyanonickelate [K 2 Ni(CN) 4 ] at pH 4. A second mixed culture consisting of Fusarium oxysporum, Scytalidium thermophilum , and Penicillium miczynski was isolated on hexacyanoferrate [K 4 Fe(CN) 6 ] also at pH 4. Both consortia were able to grow on K 4 Fe(CN) 6 as the sole source of nitrogen under acidic conditions. Growth was associated with progressive removal of cyanide from the culture supernatant. After the termination of growth, at least 50% of the total cyanide had been degraded. Growth of the fungi on K 2 Ni( 14 CN) 4 as a source of nitrogen at pH 7 yielded 14 C-labelled carbon dioxide. Growth of the Fusarium isolates on K 2 Ni(CN) 4 at pH 7, associated with the removal of cyanide, required 5 days as compared to 28 days on K 4 Fe(CN) 6 at pH 4. Cyanide uptake by the fungi on K 4 Fe(CN) 6 at pH 4 occurred simultaneously with removal of iron from the biomass-free medium. Pure cultures of F. solani and F. oxysporum were grown on K 2 Ni(CN) 4 or K 4 Fe(CN) 6 in pure culture at pH 7 or 4, respectively.

Metabolism and enzymology of cyanide/metallocyanide biodegradation by Fusarium solani under neutral and acidic conditions

A strain of Fusarium solani degrades metal-complexed cyanides under neutral and acidic pH conditions. Cyanide undergoes hydrolysis to formamide by cyanide hydratase (formamide hydrolyase, EC 4.2.1.66) which is in turn hydrolyzed to ammonia and formic acid by an amidase. The ammonia is then utilized for growth. Formic acid does not accumulate in the medium presumably due to its conversion to CO2 by formate dehydrogenase. Cyanide hydratase activity is induced by cyanide or metallocyanides under both acidic and neutral pH conditions, but not by ammonia or formamide; however, the fungus can utilize formamide as the source of nitrogen for growth. F. solani biotransforms K2Ni(CN)4 and K4Fe(CN)6 to ammonia under neutral and acidic pH conditions, respectively. The semipurified enzyme has optimal activity for KCN at pH 7.5 and has a Km of 4.7 mm and a Vmax of 1.7 μmol min−1 mg−1 protein. Enzyme purification revealed that the native molecular weight of the enzyme is greater than 300 kDa due to its elution in the void volume during gel filtration, and comprises subunits with a molecular mass of approximately 45 kDa. The n-terminal sequence of the purified cyanide hydratase has a strong homology to previously purified cyanide hydratases, nitrilases, and a cyanidase enzyme.

Electrokinetic remediation of metals and organics from historically contaminated soil

The electrokinetic remediation of an historically contaminated soil is described. The soil was contaminated with a range of metals including lead, zinc, manganese, copper and arsenic, polycyclic aromatic hydrocarbons (PAHs) and benzene, toluene, ethylbenzene and xylene (BTEX). A small-scale experiment (973.2 g dry weight soil), utilising a planar electrode configuration, investigated the potential for moving metals and organics. After 23 days treatment at a current density of 3.72 A /m−2, 44% of calcium and 29% of manganese were removed from the soil at the cathode. Of the other contaminating metals, zinc and lead moved towards the cathode, but with no significant removal from the soil. Movement of PAHs was also observed, with a 94% reduction in concentration in the third of the soil closest to the anode after 23 days. A larger scale experiment (46.7 kg dry weight soil) utilised a hexagonal array of tubular anodes surrounding a central tubular cathode. Treatment for 112 days led to acidification of the soil to pH 2.59 closest to the anode in a direct line between the anode and cathode. Soil not directly in line between the electrodes was not acidified significantly. Movement of metal ions was observed, in line with the electrodes, with concentrations of lead and arsenic increasing to 162% and 171% of starting concentrations closest to the anode, respectively, and those of zinc, copper and manganese decreasing to 42%, 68% and 57%, respectively. At positions not directly in line with the electrodes, no significant metal movements were observed. Overall, there was no significant removal of contaminating metals from the soil. PAHs and BTEX compounds were moved by electroosmosis towards the cathode, with soil concentrations of PAHs reduced from 720 mgkg−1 to 4.7 mgkg−1 after 22 days. PAHS (28 mg) and benzene (9660 mg) were recovered in granular activated carbon (GAC) columns. © 2000 Society of Chemical Industry

The effects of direct electric current on the viability and metabolism of acidophilic bacteria

Abstract The application of direct electric current to soil to remove metal ions is an emerging remediation technology. The combination of this approach with bioremediation requires that soil bacteria be viable and metabolically active under the applied currents (20 mA cm −2 ) and their imposed acidic conditions. In this study, sulfur-oxidizing bacteria (SOBs; a mixed culture and pure culture of Thiobacillus ferrooxidans ) growing on elemental sulfur, and the acidophilic heterotroph Acidiphilium SJH, growing on glucose, have been chosen as representative organisms. In liquid culture, low cell densities of T. ferrooxidans and Acidiphilium SJH were inactivated by the current; however, a high cell density of SOBs was able to recover activity when the current was switched off, and a high density of Acidiphilium SJH was able to grow despite the presence of current. In soil slurries (5, 10, and 30% w/v silt soil), indigenous SOBs were metabolically active in the presence of current; sulfate production was enhanced. There was also enhanced glucose consumption of Acidiphilium SJH in 10 and 30% slurries; however, no protective effect or increased metabolism occurred when T. ferrooxidans was introduced into soil slurries.

Identification and characterisation of bacterial populations of an in-use metal-working fluid by phenotypic and genotypic methodology

In-use metal-working fluids (MWFs) are vulnerable to microbial attack, resulting in untimely biodeterioration, thus rendering them less effective as coolants and lubricants. The aim of this study was to investigate the microbial diversity of in-use emulsifiable oil metal-working fluids, and use the information derived to develop strategies to improve metal-working fluid formulation and longevity, and to improve microbial exploitation for biological disposal routes using bioreactors. In-use MWF samples from five different metal-working machines were analysed using phenotypic, genotypic, and in situ detection methods to determine total background bacterial communities and the composition of enriched degraders. Of 66 bacterial isolates, 13 species from 8 genera were identified by fatty acid methyl ester (FAME) analysis. Genotypic (total) diversity, determined by denaturing gradient gel electrophoresis (DGGE), reflected the low diversity shown by FAME analysis. Both DGGE and in situ microscopy (fluorescent in situ hybridisation, FISH) revealed highly conserved microbial communities in MWF from different machines and applications.

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.

Bioaugmentation strategies for remediating mixed chemical effluents.

Operationally exhausted metal working fluids are chemically mixed, produced in large quantities (400 000 tonnes year in the U.K.), and potentially environmentally toxic. It is essential to develop more reliable and economical approaches for their disposal. We investigated the effectiveness of a defined bacterial consortium, constructed specifically for treating metal‐working fluid (MWF), and contrasted its performance to that of undefined inocula from activated sludge. Construction of the consortium was based on knowledge of the diversity of bacterial communities that naturally colonize MWF and determination of their catabolic abilities and tolerance to the chemical constituents. Chemical analysis of the inoculated MWF bioreactor revealed that, after 100 h at 28 °C, the defined inoculum reduced the pollution load by over 80% from an initial chemical oxygen demand of approximately 48 000 mg L‐1. The inocula performance was approximately 50% more effective than that of the undefined microbial community from the activated sludge. Furthermore, the performance of the constructed consortium was more reproducible than that of an undefined community, an essential feature for bioaugmentation treatment of industrial wastes.

Conversion of 3-cyanopyridine to nicotinic acid by Nocardia rhodochrous LL100-21

Abstract 3-Cyanopyridinase activity, i.e. the ability to convert 3-cyanopyridine to nicotinic acid plus ammonia, was induced in stationary phase cultures of Nocardia rhodochrous LL100-21 by the addition of 2-, 3-, or 4-cyanopyridine or benzonitrile; the latter nitrile gave maximum induction. Harvested bacteria possessing 3-cyanopyridinase activity could stoichiometrically convert 3-cyanopyridine at concentrations of up to 0.5 m to nicotinic acid. Both 3-cyanopyridine and nicotinic acid inhibited the hydrolysis of 3-cyanopyridine by intact bacteria. Bacteria immobilized in calcium alginate beads and used in column bioreactors retained 3-cyanopyridinase activity for over 150 h when continuously supplied with 0.3 m 3-cyanopyridine.

Degradation of copper-NTA by Mesorhizobium sp. NCIMB 13524

Abstract In a series of enrichment isolations, a mixed culture was obtained from soil which was able to degrade copper nitrilotriacetate. In phosphate buffered (metal-complexing) medium this mixed culture grew on and completely degraded Mn-, Fe-, Co-, Cu- and Zn-NTA after 14 d incubation at 30°C. In PIPES-buffered (non-complexing) medium, the mixed culture completely degraded free NTA and Mn-, Fe- and Zn-NTA; Cu-NTA was partially degraded (54%) and Co-NTA was not degraded. A pure culture of Mesorhizobium sp. (NCIMB 13524) isolated from the mixed culture completely degraded free NTA and Mn-, Fe-, Co-, Ni- Cu-, and Zn-NTA in phosphate buffer after 2– 24 d incubation at 30°C. In PIPES buffer, growth and NTA degradation by Mesorhizobium sp. was inhibited stoichiometrically by copper, suggesting that only the free NTA was degraded. In addition, fully complexed Ni-NTA was not degraded in PIPES buffer, and Co-NTA was only partially degraded (18.1%). The importance of speciation, and the effect of the buffer on NTA degradation is discussed.

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

Abstract. 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.