Bioreactor microbial ecosystems for thiocyanate and cyanide degradation unravelled with genome-resolved metagenomics.

Gold ore processing uses cyanide (CN(-) ), which often results in large volumes of thiocyanate- (SCN(-) ) contaminated wastewater requiring treatment. Microbial communities can degrade SCN(-) and CN(-) , but little is known about their membership and metabolic potential. Microbial-based remediation strategies will benefit from an ecological understanding of organisms involved in the breakdown of SCN(-) and CN(-) into sulfur, carbon and nitrogen compounds. We performed metagenomic analysis of samples from two laboratory-scale bioreactors used to study SCN(-) and CN(-) degradation. Community analysis revealed the dominance of Thiobacillus spp., whose genomes harbour a previously unreported operon for SCN(-) degradation. Genome-based metabolic predictions suggest that a large portion of each bioreactor community is autotrophic, relying not on molasses in reactor feed but using energy gained from oxidation of sulfur compounds produced during SCN(-) degradation. Heterotrophs, including a bacterium from a previously uncharacterized phylum, compose a smaller portion of the reactor community. Predation by phage and eukaryotes is predicted to affect community dynamics. Genes for ammonium oxidation and denitrification were detected, indicating the potential for nitrogen removal, as required for complete remediation of wastewater. These findings suggest optimization strategies for reactor design, such as improved aerobic/anaerobic partitioning and elimination of organic carbon from reactor feed.

The effect of degree and timing of nitrogen limitation on lipid productivity in Chlorella vulgaris.

Improvements in lipid productivity would enhance the economic feasibility of microalgal biodiesel. In order to optimise lipid productivity, both the growth rate and lipid content of algal cells must be maximised. The lipid content of many microalgae can be enhanced through nitrogen limitation, but at the expense of biomass productivity. This suggests that a two-stage nitrogen supply strategy might improve lipid productivity. Two different nitrogen supply strategies were investigated for their effect on lipid productivity in Chlorella vulgaris. The first was an initial nitrogen-replete stage, designed to optimise biomass productivity, followed by nitrogen limitation to enhance lipid content (two-stage batch) and the second was an initial nitrogen-limited stage, designed to maximise lipid content, followed by addition of nitrogen to enhance biomass concentration (fed-batch). Volumetric lipid yield in nitrogen-limited two-stage batch and fed-batch was compared with that achieved in nitrogen-replete and nitrogen-limited batch culture. In a previous work, maximum lipid productivity in batch culture was found at an intermediate level of nitrogen limitation (starting nitrate concentration of 170 mg L(-1)). Overall lipid productivity was not improved by using fed-batch or two-stage culture strategies, although these strategies showed higher volumetric lipid concentrations than nitrogen-replete batch culture. The dilution of cultures prior to nitrogen deprivation led to increased lipid accumulation, indicating that the availability of light influenced the rate of lipid accumulation. However, dilution did not lead to increased lipid productivity due to the resulting lower biomass concentration.

The effect of nitrogen limitation on lipid productivity and cell composition in Chlorella vulgaris.

Chlorella vulgaris accumulates lipid under nitrogen limitation, but at the expense of biomass productivity. Due to this tradeoff, improved lipid productivity may be compromised, despite higher lipid content. To determine the optimal degree of nitrogen limitation for lipid productivity, batch cultures of C. vulgaris were grown at different nitrate concentrations. The growth rate, lipid content, lipid productivity and biochemical and elemental composition of the cultures were monitored for 20 days. A starting nitrate concentration of 170 mg L(-1) provided the optimal tradeoff between biomass and lipid production under the experimental conditions. Volumetric lipid yield (in milligram lipid per liter algal culture) was more than double that under nitrogen-replete conditions. Interpolation of the data indicated that the highest volumetric lipid concentration and lipid productivity would occur at nitrate concentrations of 305 and 241 mg L(-1), respectively. There was a strong correlation between the nitrogen content of the cells and the pigment, protein and lipid content, as well as biomass and lipid productivity. Knowledge of the relationships between cell nitrogen content, growth, and cell composition assists in the prediction of the nitrogen regime required for optimal productivity in batch or continuous culture. In addition to enhancing lipid productivity, nitrogen limitation improves the lipid profile for biodiesel production and reduces the requirement for nitrogen fertilizers, resulting in cost and energy savings and a reduction in the environmental burden of the process.

Attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum cultured under varying conditions to pyrite, chalcopyrite, low-grade ore and quartz in a packed column reactor.

The attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum spp. grown on ferrous medium or adapted to a pyrite mineral concentrate to four mineral substrata, namely, chalcopyrite and pyrite concentrates, a low-grade chalcopyrite ore (0.5 wt%) and quartzite, was investigated. The quartzite represented a typical gangue mineral and served as a control. The attachment studies were carried out in a novel particle-coated column reactor. The saturated reactor containing glass beads, which were coated with fine mineral concentrates, provided a quantifiable surface area of mineral concentrate and maintained good fluid flow. A. ferrooxidans and Leptospirillum spp. had similar attachment characteristics. Enhanced attachment efficiency occurred with bacteria grown on sulphide minerals relative to those grown on ferrous sulphate in an ore-free environment. Selective attachment to sulphide minerals relative to gangue materials occurred, with mineral adapted cultures attaching to the minerals more efficiently than ferrous grown cultures. Mineral-adapted cultures showed highest levels of attachment to pyrite (74% and 79% attachment for A. ferrooxidans and L. ferriphilum, respectively). This was followed by attachment of mineral-adapted cultures to chalcopyrite (63% and 58% for A. ferrooxidans and L. ferriphilum, respectively). A. ferrooxidans and L. ferriphilum exhibited lower levels of attachment to low-grade ore and quartz relative to the sulphide minerals.

Effect of culture conditions on the competitive interaction between lactate oxidizers and fermenters in a biological sulfate reduction system.

Kinetic constants (µ(max) and K(s)) describing the predominance of lactate oxidation and fermentation were determined in chemostat cultures. The kinetics of sulfate reduction and lactate utilization were determined from 0.5 to 5d residence times at feed sulfate concentrations of 1.0-10.0 g l(-1). The kinetics of lactate fermentation in the absence of sulfate were investigated at residence times of 0.5-5d. The lactate oxidizers (LO) were characterized by a µ(max) of 0.2h(-1) and K(s) value of 0.6 g l(-1) compared with a µ(max) of 0.3h(-1) and K(s) of 3.3 g l(-1) for the lactate fermenters (LF). Using mathematical models, it was shown that LO competed more effectively for lactate at low lactate concentrations (≤5 g l(-1)) and high sulfide concentrations (0.5 g l(-1)). Lactate fermenters outcompeted the oxidizers under conditions of excess lactate (>5 g l(-1)) and low sulfide (0.014-0.088 g l(-1)).

Interference by pigment in the estimation of microalgal biomass concentration by optical density.

Optical density is used as a convenient indirect measurement of biomass concentration in microbial cell suspensions. Absorbance of light by a suspension can be related directly to cell density using a suitable standard curve. However, inaccuracies can be introduced when the pigment content of the cells changes. Under the culture conditions used, pigment content of the microalga Chlorella vulgaris varied between 0.5 and 5.5% of dry weight with age and culture conditions. This led to significant errors in biomass quantification over the course of a growth cycle, due to the change in absorbance. Using a standard curve generated at a single time point in the growth cycle to calculate dry weight (dw) from optical density led to average relative errors across the growth cycle, relative to actual dw, of between 9 and 18% at 680 nm and 5 and 13% at 750 nm. When a standard curve generated under low pigment conditions was used to estimate biomass under normal pigment conditions, average relative errors in biomass estimation relative to actual dw across the growth cycle were 52% at 680 nm and 25% at 750 nm. Similar results were found with Scenedesmus, Spirulina and Nannochloropsis. Suggested strategies to minimise error include selection of a wavelength that minimises absorbance by the pigment, e.g. 750 nm where chlorophyll is the dominant pigment, and generation of a standard curve towards the middle, or across the entire, growth cycle.

Study of anaerobic lactate metabolism under biosulfidogenic conditions.

Biological sulfate reduction (BSR) has been reported to have potential for the treatment of acid mine drainage (AMD). The provision of a suitable carbon source and electron donor for this process remains a challenge. Lactate offers potential advantages as carbon source and electron donor in the biological sulfate reduction process. As this substrate is utilized by both fermentative bacteria and oxidative sulfate-reducing bacteria (SRB), the effect of feed sulfate concentration on the lactate pathways utilized under biosulfidogenic conditions was investigated. Studies were carried out in chemostat bioreactors across a range of residence times, using an enriched culture of SRB. The stoichiometry of biological sulfate reduction was affected by feed sulfate concentration and dilution rate. Incomplete oxidation of lactate was dominant at low feed sulfate concentration (1.0 g/L), while the yield of propionate from lactate metabolism increased at feed sulfate concentrations of 2.5-10.0 g/L, indicating the occurrence of lactate fermentation. Furthermore, at each sulfate feed concentration, in the range 2.5-10.0 g/L, the ratio in which lactate was metabolized by the oxidative and fermentative pathways varied with varying dilution rates. Lactate oxidation was higher at a feed sulfate concentration of 10.0 g/L relative to 2.5 and 5.0 g/L. The volumetric lactate utilization rate was enhanced by increasing the feed sulfate concentration. However, the proportion of total lactate consumed that was channelled into providing electrons for other activities apart from sulfate reduction also increased over the range of increasing sulfate concentrations studied and appeared to be a function of residual lactate and sulfide concentrations.