In vitro toxicity assessment of fungal-synthesized cadmium sulfide quantum dots using bacteria and seed germination models.

There is currently controversy over the use of quantum dots (QDs) in biological applications due to their toxic effects. Therefore, the purpose of the present study was to evaluate the toxic effect of chemical and biogenic (synthesized by Fusarium oxysporum f. sp. lycopersici) cadmium sulfide quantum dots (CdSQDs) using a bacterial model of Escherichia coli and sprouts of Lactuca sativa L. with the aim to foresee its use in the near future in biological systems. Physicochemical properties of both types of CdSQDs were determined by TEM, XRD, zeta potential and fluorescence spectroscopy. Both biogenic and chemical CdSQDs showed agglomerates of spherical CdSQDs with diameters of 4.14 nm and 3.2 nm, respectively. The fluorescence analysis showed a band around 361 nm in both CdSQDs, the zeta potential was -1.81 mV for the biogenic CdSQDs and -5.85 mv for the chemical CdSQDs. Results showed that chemical CdSQDs, presented inhibition in the proliferation of E. coli cell in a dose-dependent manner, unlike biogenic CdSQDs, that only at its highest concentration showed an antibacterial activity. Also, it was observed that after incubation with chemical and biogenic CdSQDs of L. sativa L. seeds, only the biogenic CdSQDs showed no inhibition on seed germination. In summary, our results suggest that the production route has a significant effect on the toxicity of QDs; in addition, it seems that the biological coating of the CdSQDs from F. oxysporum f. sp. lycopersici inhibit their toxic effect on bacterial strains and plant seeds.

Water dispersible ZnSe/ZnS quantum dots: Assessment of cellular integration, toxicity and bio-distribution.

Quantum dots (QDs) comprise an emerging group of materials with innumerable number of possibilities in biological research including cellular labelling. Among the leading members in this category, ZnSe/ZnS quantum dots (QDs) hold greater attractive possibilities in imaging primarily due to their higher biocompatibility and dispersibility. Nevertheless, the inherent toxicity of ZnSe/ZnS QDs is not yet completely explored which largely compromise most of their biomedical application potential. Strong blue emitting water soluble QDs effectively synthesized by aqueous phase route. Synthesized QDs further subjected to various optical and physicochemical characterization. Approximately 5-6 nm sized ZnSe/ZnS QDs illuminated bluish green fluorescence under UV lamp. Present study addresses possible adverse effects of ZnSe/ZnS QDs in hepatic system using HepG2 cells; which is the routinely employed in vitroliver cell model. A bundle of assays wasperformed out to reveal the cytotoxic nature of ZnSe/ZnS QDs and the mechanism behind it. Herein, absorption, distribution, metabolism, excretion and toxicity (ADME and T) of ZnSe/ZnS in mice were profiled in detail followed by intravenous (i.v.) and intraperitoneal (i.p.) administration at a dose of 10 mg/kg body weight. In a short review, it could be state that ZnSe/ZnS QDs did not exhibit any significant in vivo toxicity outcome in mice.

Realizing a high-rate sulfidogenic reactor driven by sulfur-reducing bacteria with organic substrate dosage minimization and cost-effectiveness maximization.

Biological sulfur reduction is an attractive sulfidogenic technology for the treatment of organics-deficient metal-laden wastewater, because it theoretically reduces the electron donor consumption by 75%, compared to sulfate reduction. However, reducing the external organic substrate dosage may lower the sulfur reduction rate. Supplying with a more biodegradable organic substrate could possibly enhance sulfidogenic activity but also increase the chemical cost. Therefore, the sulfide production performance of a sulfur-reducing bioreactor feeding with varied levels of organic supply, and different types of organic substrates were investigated. The results showed that high-rate sulfide production (12.30 mg S/L/h) in a sulfur-reducing bioreactor can be achieved at the minimal dosage of organic substrate as low as 39 mg C/L of organic carbon in the influent. Changing the type of organic substrate posed a significant effect on the sulfidogenic activity in the sulfur-reducing bioreactor. Sodium acetate was found to be the optimal substrate to achieve the highest sulfide production rate (28.20 mg S/L/h) by sulfur-reducing bacteria (S0RB), followed by ethanol, methanol, glycerol, pyruvic acid, acetic acid, glucose, sucrose, malic acid, sodium formate, formic acid, N-propanol, N-butanol, lactic acid, sodium lactate, propionic acid and sodium propionate (2.87 mg S/L/h as the lowest rate). However, the cost-effectiveness analysis showed that glucose was the most cost-effective organic substrate to realize the sulfur reduction process in high sulfide production rate (20.13 mg S/L/h) and low chemical cost (5.94 kg S/$). The utilization pathway of the different organic substrates in the sulfur-reducing bioreactor was also discussed.

Design and shelf stability assessment of bacterial agents for simultaneous removal of methane and odors.

Two types of solid bacterial agents for the simultaneous removal of methane and odor were designed using humic soil (De-MO-1) and the mixture of humic soil and tobermolite (De-MO-2) as biocarriers. The bacterial consortium, having the removability of methane and dimethyl sulfide (DMS), was immobilized in the biocarriers, and then stored at room temperature for 375 days without additional treatment. Although the lag period, of which the incubation time required for removing methane and DMS, tended to increase over the storage period, the removability of methane and DMS was maintained during 375 days in both bacterial agents. Key bacteria associated with the removal of methane and odors (Streptomyces, Promicromonospora, Paracoccus, Lysobacter, Sphingopyxis and Methylosystis) could keep their abundance during the storage period. The richness and evenness values of the bacterial communities in De-MO-1 and De-MO-2 ranged 4.89 ∼ 6.50 and 0.89 ∼ 0.98, respectively, indicating that high bacterial diversity was maintained during the storage period. The results suggest that De-MO-1 and De-MO-2, designed for the simultaneous removal of methane and odors, had shelf stabilities over one year.

A metatranscriptomics-based assessment of small-scale mixing of sulfidic and oxic waters on redoxcline prokaryotic communities.

Lateral intrusions of oxygen caused by small-scale mixing are thought to shape microbial activity in marine redoxclines. To examine the response of prokaryotes to such mixing events we employed a shipboard mixing experiment in the euxinic central Baltic Sea: oxic, nitrate containing and sulfidic water samples without detectable oxygenized substances were incubated directly or after mixing. While nitrate, nitrite and ammonium concentrations stayed approximately constant in all incubations, we observed a decrease of sulfide after the contact with oxygen in the sulfide containing incubations. The transcription of marker genes from chemolithoauthotrophic key players including archaeal nitrifiers as well as gammaproteobacterial and campylobacterial autotrophic organisms that couple denitrification with sulfur-oxidation were followed at four time points within 8.5 h. The temporally contrasting transcriptional profiles of gammaproteobacterial and campylobacterial denitrifiers that depend on the same inorganic substrates pointed to a niche separation. Particular archaeal and campylobacterial marker genes involved in nitrification, denitrification and sulfur oxidation, which depend on oxidized substrates, were highly upregulated in the anaerobic sulfidic samples. We suggest that, despite the absence of measurable oxygenated compounds in the sulfidic water, frequent intermittent small-scale intrusions stimulate the permanent upregulation of genes involved in nitrification, denitrification and sulfur oxidation.

Assessment of sulfide production in a full scale wastewater sludge rising main.

Production and build-up of sulfide in wastewater systems, especially downstream of rising mains, may lead to severe odour nuisance, toxic environments and high risk of corrosion. Due to increased population migration towards cities and lower area availability for treatment facilities, rising mains for the conveyance of wastewater sludge are becoming more frequent, and research on sulfide build-up in such cases is needed. In this paper the findings of the work carried out in a full scale wastewater sludge rising main, operated during different seasons and under distinct conditions are presented (comprising both the start-up and normal operation stages of the facility). Results showed a sulfide build-up rate of 3.24 g S-2 m-2 d-1 in the summer and of 2.30 g S-2 m-2 d-1 during the winter. The ratio of sulfate reduction to sulfide production (SO4-2/S-2) was of roughly 3 to 1, as expected. Furthermore, obtained results allowed adjusting a second order polynomial empirical equation for the forecasting of sulfide build-up within the sludge rising main. The obtained equation for sludge significantly differs from existing equations obtained for wastewater. Moreover, this work also allowed obtaining new insight into the positive influence of biofilm and hydraulic retention time in the biological sulfide generation, as well as into its variation along the length of the rising main.

Assessment of biofilm changes and concentration-depth profiles during arsenopyrite oxidation by Acidithiobacillus thiooxidans.

Biofilm formation and evolution are key factors to consider to better understand the kinetics of arsenopyrite biooxidation. Chemical and surface analyses were carried out using Raman spectroscopy, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), glow discharge spectroscopy (GDS), and protein analysis (i.e., quantification) in order to evaluate the formation of intermediate secondary compounds and any significant changes arising in the biofilm structure of Acidithiobacillus thiooxidans during a 120-h period of biooxidation. Results show that the biofilm first evolves from a low cell density structure (1 to 12 h) into a formation of microcolonies (24 to 120 h) and then finally becomes enclosed by a secondary compound matrix that includes pyrite (FeS2)-like, S n2-/S0, and As2S3 compounds, as shown by Raman and SEM-EDS. GDS analyses (concentration-depth profiles, i.e., 12 h) indicate significant differences for depth speciation between abiotic control and biooxidized surfaces, thus providing a quantitative assessment of surface-bulk changes across samples (i.e. reactivity and /or structure-activity relationship). Respectively, quantitative protein analyses and CLSM analyses suggest variations in the type of extracellular protein expressed and changes in the biofilm structure from hydrophilic (i.e., exopolysaccharides) to hydrophobic (i.e., lipids) due to arsenopyrite and cell interactions during the 120-h period of biooxidation. We suggest feasible environmental and industrial implications for arsenopyrite biooxidation based on the findings of this study.

Model-Based Feasibility Assessment of Membrane Biofilm Reactor to Achieve Simultaneous Ammonium, Dissolved Methane, and Sulfide Removal from Anaerobic Digestion Liquor.

In this study, the membrane biofilm reactor (MBfR) is proposed to achieve simultaneous removal of ammonium, dissolved methane, and sulfide from main-stream and side-stream anaerobic digestion liquors. To avoid dissolved methane stripping, oxygen is introduced through gas-permeable membranes, which also from the substratum for the growth of a biofilm likely comprising ammonium oxidizing bacteria (AOB), anaerobic ammonium oxidation (Anammox) bacteria, denitrifying anaerobic methane oxidation (DAMO) microorganisms, aerobic methane oxidizing bacteria (MOB), and sulfur oxidizing bacteria (SOB). A mathematical model is developed and applied to assess the feasibility of such a system and the associated microbial community structure under different operational conditions. The simulation studies demonstrate the feasibility of achieving high-level (>97.0%), simultaneous removal of ammonium, dissolved methane, and sulfide in the MBfRs from both main-stream and side-stream anaerobic digestion liquors through adjusting the influent surface loading (or hydraulic retention time (HRT)) and the oxygen surface loading. The optimal HRT was found to be inversely proportional to the corresponding oxygen surface loading. Under the optimal operational conditions, AOB, DAMO bacteria, MOB, and SOB dominate the biofilm of the main-stream MBfR, while AOB, Anammox bacteria, DAMO bacteria, and SOB coexist in the side-stream MBfR to remove ammonium, dissolved methane, and sulfide simultaneously.

Cystathionine ß-synthase-derived hydrogen sulfide is involved in human malignant hyperthermia.

Hydrogen sulfide is an endogenous gasotransmitter and its mechanism of action involves activation of ATP-sensitive K(+) channels and phosphodiesterase inhibition. As both mechanisms are potentially involved in malignant hyperthermia (MH), in the present study we addressed the involvement of the L-cysteine/hydrogen sulfide pathway in MH. Skeletal muscle biopsies obtained from 25 MH-susceptible (MHS) and 56 MH-negative (MHN) individuals have been used to perform the in vitro contracture test (IVCT). Quantitative real-time PCR (qPCR) and Western blotting studies have also been performed. Hydrogen sulfide levels are measured in both tissue samples and plasma. In MHS biopsies an increase in cystathionine ß-synthase (CBS) occurs, as both mRNA and protein expression compared with MHN biopsies. Hydrogen sulfide biosynthesis is increased in MHS biopsies (0.128±0.12 compared with 0.943±0.13 nmol/mg of protein per min for MHN and MHS biopsies, respectively; P<0.01). Addition of sodium hydrosulfide (NaHS) to MHS samples evokes a response similar, in the IVCT, to that elicited by either caffeine or halothane. Incubation of MHN biopsies with NaHS, before caffeine or halothane challenge, switches an MHN to an MHS response. In conclusion we demonstrate the involvement of the L-cysteine/hydrogen sulfide pathway in MH, giving new insight into MH molecular mechanisms. This finding has potential implications for clinical care and could help to define less invasive diagnostic procedures.

Pyrosequencing-Based Assessment of the Microbial Community Structure of Pastoruri Glacier Area (Huascarán National Park, Perú), a Natural Extreme Acidic Environment.

The exposure of fresh sulfide-rich lithologies by the retracement of the Nevado Pastoruri glacier (Central Andes, Perú) is increasing the presence of heavy metals in the water as well as decreasing the pH, producing an acid rock drainage (ARD) process in the area. We describe the microbial communities of an extreme ARD site in Huascarán National Park as well as their correlation with the water physicochemistry. Microbial biodiversity was analyzed by FLX 454 sequencing of the 16S rRNA gene. The suggested geomicrobiological model of the area distinguishes three different zones. The proglacial zone is located in the upper part of the valley, where the ARD process is not evident yet. Most of the OTUs detected in this area were related to sequences associated with cold environments (i.e., psychrotolerant species of Cyanobacteria or Bacteroidetes). After the proglacial area, an ARD-influenced zone appeared, characterized by the presence of phylotypes related to acidophiles (Acidiphilium) as well as other species related to acidic and cold environments (i.e., acidophilic species of Chloroflexi, Clostridium and Verrumicrobia). Sulfur- and iron-oxidizing acidophilic bacteria (Acidithiobacillus) were also identified. The post-ARD area was characterized by the presence of OTUs related to microorganisms detected in soils, permafrost, high mountain environments, and deglaciation areas (Sphingomonadales, Caulobacter or Comamonadaceae).

Assessment of pH shock as a method for controlling sulfide and methane formation in pressure main sewer systems.

Caustic dosing to raise pH above 10.0 for short periods (hours) is often used by water utilities for controlling sulfide formation in sewers. However the effectiveness of this strategy is rarely reported and the impact of pH level and exposure time on the effectiveness is largely unknown. The effectiveness of this strategy under various pH levels (10.5-12.5) and exposure time (0.5-6.0 h) in controlling sulfide and methane production was evaluated in laboratory scale anaerobic sewer reactors and then in a real sewer system. Laboratory studies showed that the sulfide production rate of the laboratory sewer biofilm was reduced by 70-90% upon the completion of the pH shock, while the methane production rate decreased by 95-100%. It took approximately one week for the sulfate-reducing activity to recover to normal levels. In comparison, the methanogenic activities recovered to only about 10% in 4 weeks. The slow recovery is explained by the substantially loss of cell viability upon pH shocks, which recovered slowly after the shocks. Laboratory studies further revealed that a pH level of 10.5 for 1-2 h represent cost-effective conditions for the pH shock treatment. However, field trials showed a higher pH (11.5) and larger dosing times are needed due to the pH decreases along the sewer line and at the two ends of the caustic-receiving wastewater slugs due to dilution. To have effective sulfide and methane control, it is important to ensure effective conditions (pH > 10.5 and duration >1-2 h) for the entire sewer line.

Scalable economic extracellular synthesis of CdS nanostructured particles by a non-pathogenic thermophile.

We report microbially facilitated synthesis of cadmium sulfide (CdS) nanostructured particles (NP) using anaerobic, metal-reducing Thermoanaerobacter sp. The extracellular CdS crystallites were <10 nm in size with yields of ~3 g/L of growth medium/month with demonstrated reproducibility and scalability up to 24 L. During synthesis, Thermoanaerobacter cultures reduced thiosulfate and sulfite salts to H2S, which reacted with Cd²âº cations to produce thermodynamically favored NP in a single step at 65 °C with catalytic nucleation on the cell surfaces. Photoluminescence (PL) analysis of dry CdS NP revealed an exciton-dominated PL peak at 440 nm, having a narrow full width at half maximum of 10 nm. A PL spectrum of CdS NP produced by dissimilatory sulfur reducing bacteria was dominated by features associated with radiative exciton relaxation at the surface. High reproducibility of CdS NP PL features important for scale-up conditions was confirmed from test tubes to 24 L batches at a small fraction of the manufacturing cost associated with conventional inorganic NP production processes.

Complete genome sequence of the hyperthermophilic archaeon Thermococcus sp. strain AM4, capable of organotrophic growth and growth at the expense of hydrogenogenic or sulfidogenic oxidation of carbon monoxide.

Analysis of the complete genome of Thermococcus sp. strain AM4, which was the first lithotrophic Thermococcales isolate described and the first archaeal isolate to exhibit a capacity for hydrogenogenic carboxydotrophy, reveals a proximity with Thermococcus gammatolerans, corresponding to close but distinct species that differ significantly in their lithotrophic capacities.

Assessment of the solubility and bioaccessibility of arsenic in realgar wine using a simulated gastrointestinal system.

Consumption of arsenic (As) wine is a traditional activity during the classic Chinese festival of Duanwu, colloquially known worldwide as the Dragon Boat Day. Arsenic wine is drunk on the morning of the fifth day of the fifth lunar calendar month to commemorate the death of Qu Yuan, a famed Chinese poet who drowned himself in protest of a corrupt government, and to protect against ill fortune. Although realgar minerals are characteristically composed of sparingly soluble tetra-arsenic tetra-sulfides (As(4)S(4)), purity does vary with up to 10% of As being present as non-sulfur bound species, such as arsenate (As(V)) and arsenite (As(III)). Despite, the renewed interest in As speciation and the bioaccessibility of the active As components in realgar based Chinese medicines, little is known about the safety surrounding the cultural practice of drinking As wine. In a series of experiments the speciation and solubility of As in a range of wines were investigated. Furthermore, a simulated gastrointestinal system was employed to predict the impact of digestive processes on As bioavailability. The predominant soluble As species found in all the wines were As(III) and As(V). Based on typical As wine recipes employing 0.1 g realgar mL(-1) wine, the concentration of dissolved As ranged from ca. 100 to 400 mg L(-1) depending on the ethanol content of the preparation: with the As solubility found to be higher in wines with a lower proportion of ethanol. Based on a common 100 mL measure of wine with a concentration of 400 mg As L(-1), the amount of soluble As would equate to around half of the acute minimal lethal dose for adults. This is likely an underestimate of the bioaccessible concentration, as a three-fold increase in bioaccessibility could be observed in the intestinal phase based on the results from the stimulated gastrointestinal system.

Metalloproteins and the pyrite-based origin of life: a critical assessment.

We critically examine the proposal by Wächtershäuser (Prokaryotes 1:275-283, 2006a, Philos Trans R Soc Lond B Biol Sci 361: 787-1808, 2006b) that putative transition metal binding sites in protein components of the translation machinery of hyperthermophiles provide evidence of a direct relationship with the FeS clusters of pyrite and thus indicate an autotrophic origin of life in volcanic environments. Analysis of completely sequenced cellular genomes of Bacteria, Archaea and Eucarya does not support the suggestion by Wächtershäuser (Prokaryotes 1:275-283, 2006a, Philos Trans R Soc Lond B Biol Sci 361: 787-1808, 2006b) that aminoacyl-tRNA synthetases and ribosomal proteins bear sequence signatures typical of strong covalent metal bonding whose absence in mesophilic species reveals a process of adaptation towards less extreme environments.

Production-related petroleum microbiology: progress and prospects.

Microbial activity in oil reservoirs is common. Methanogenic consortia hydrolyze low molecular weight components to methane and CO2, transforming light oil to heavy oil to bitumen. The presence of sulfate in injection water causes sulfate-reducing bacteria to produce sulfide. This souring can be reversed by nitrate, stimulating nitrate-reducing bacteria. Removing biogenic sulfide is important, because it contributes to pitting corrosion and resulting pipeline failures. Increased water production eventually makes oil production uneconomic. Microbial fermentation products can lower oil viscosity or interfacial tension and produced biomass can block undesired flow paths to produce more oil. These biotechnologies benefit from increased understanding of reservoir microbial ecology through new sequence technologies and help to decrease the environmental impact of oil production.

A laboratory assessment of the impact of brewery wastewater discharge on sulfide and methane production in a sewer.

The impact of brewery wastewater discharge on sulfide and methane production in a sewer was assessed. Experiments were carried out on laboratory scale sewer reactors consisting of both an experimental and a control reactor. The control reactor was intermittently fed with real fresh sewage while the experimental reactor was fed with a mixture of brewery and domestic wastewater at two different proportions (10 and 25% v/v). 10% v/v discharge of brewery wastewater increased the H2S and CH4 production rates in the sewer reactor by 40% and 30%, respectively. When the brewery wastewater fraction was increased to 25% v/v, the H2S production rate of the experimental reactor decreased to the level of the control reactor. In contrast, the CH4 production rate maintained at a level that was 30% higher than that in the control reactor. These results indicate that the discharge of brewery wastewater into sewers can give negative impacts in relation to odour and corrosion management of the systems and will increase the greenhouse gas emissions from sewers. The study also reveals that the impact of trade waste on the biological reactions in sewers is complex, and requires careful experimental assessment in each case.

Decreasing sulfide generation in sewage by dosing formaldehyde and its derivatives under anaerobic conditions.

Hydrogen sulfide emission in sewers is associated with toxicity, corrosion, odour nuisance and high costs. In this study, a new method to inhibit sulfide generation by means of formaldehyde and its derivatives has been evaluated under anaerobic conditions. The possible impact of formaldehyde on an activated sludge system and an appraisal of the economic aspects are presented as well. A dosage of 19 mg L(-1) formaldehyde resulted in a decrease of the sulfide production of 90%. Dosing of 32 mg L(-1) paraformaldehyde and addition of 111 mg L(-1) ureaformaldehyde were not sufficient to inhibit the sulfide generation in sewage to the same extent. The impacts of 19 mg L(-1) formaldehyde on activated sludge, in terms of COD removal, nitrification rate and oxygen uptake rates, were negligible. This suggests that formaldehyde dosage is a feasible technique to abate the sulfide problem in sewers.

Microbial conversion of sulfur dioxide in flue gas to sulfide using bulk drug industry wastewater as an organic source by mixed cultures of sulfate reducing bacteria.

Mixed cultures of sulfate reducing bacteria (SRB) were isolated from anaerobic cultures and enriched with SRB media. Studies on batch and continuous reactors for the removal of SO(2) with bulk drug industry wastewater as an organic source using isolated mixed cultures of SRB revealed that isolation and enrichment methodology adopted in the present study were apt to suppress the undesirable growth of anaerobic bacteria other than SRB. Studies on anaerobic reactors showed that process was sustainable at COD/S ratio of 2.2 and above with optimum sulfur loading rate (SLR) of 5.46kgS/(m(3)day), organic loading rate (OLR) of 12.63kg COD/(m(3)day) and at hydraulic residence time (HRT) of 8h. Free sulfide (FS) concentration in the range of 300-390mgFS/l was found to be inhibitory to mixed cultures of SRB used in the present studies.

Characterization and reactivity assessment of organic substrates for sulphate-reducing bacteria in acid mine drainage treatment.

Acid mine drainage (AMD), which contains high concentrations of sulphate and dissolved metals, is a serious environmental problem. It can be treated in situ by sulphate reducing bacteria (SRB), but effectiveness of the treatment process depends on the organic substrate chosen to supply the bacteria's carbon source. Six natural organic materials were characterized in order to investigate how well these promote sulphate reduction and metal precipitation by SRB. Maple wood chips, sphagnum peat moss, leaf compost, conifer compost, poultry manure and conifer sawdust were investigated in terms of their carbon (TOC, TIC, DOC) and nitrogen (TKN) content, as well as their easily available substances content (EAS). Single substrates, ethanol, a mixture of leaf compost (30% w/w), poultry manure (18% w/w), and maple wood chips (2% w/w), and the same mixture spiked with formaldehyde were then tested in a 70-day batch experiment to evaluate their performance in sulphate reduction and metal removal from synthetic AMD. Metal removal efficiency in batch reactors was as high as 100% for Fe, 99% for Mn, 99% for Cd, 99% for Ni, and 94% for Zn depending on reactive mixtures. Early metal removal (0-12d) was attributed to the precipitation of (oxy)hydroxides and carbonate minerals. The lowest metal and sulphate removal efficiency was found in the reactor containing poultry manure as the single carbon source despite its high DOC and EAS content. The mixture of organic materials was most effective in promoting sulphate reduction, followed by ethanol and maple wood chips, and single natural organic substrates generally showed low reactivity. Formaldehyde (0.015% (w/v)) provided only temporary bacterial inhibition. Although characterization of substrates on an individual basis provided insight on their chemical make-up, it did not give a clear indication of their ability to promote sulphate reduction and metal removal.