Robert P. van Hille

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.

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.

Dynamic Evolution of the Microbial Community in BIOX Leaching Tanks

Laboratory scale (7 L) reactors, inoculated with the L. ferriphilum dominated BIOX inoculum, were used to test the stability of the community under controlled conditions. Further, the effect of increased temperature, solids loading and pH fluctuations on the bioleaching performance and community structure were studied. Both performance and community structure remained stable under controlled conditions (41.5°C, 20% solids loading, 7 day residence time). Increasing the solids loading to 31% did not significantly affect performance or community structure. An increase in temperature (2°C every 10 days) did not have a significant effect up to 48°C, but the increase from 48°C to 50°C resulted in the loss of L. ferriphilum and a decrease in leaching performance. A more gradual increase (1°C increments) from 48°C to 50°C resulted in a stable community, dominated by Ac. cupricumulans and Acidithiobacillus caldus. A similar shift in community structure was observed when the pH fell below pH 0.8, but this was transient and L. ferriphilum recovered dominance upon adjustment to a pH > 1.0. A further increase in temperature to 52°C resulted in the loss of At. caldus and the emergence of Sulfobacilli. However, leaching performance under these conditions was poor, despite the presence of over 10 g/L ferric iron. In addition, yeast extract was required to maintain high cell numbers at 52°C. This work has identified a selection of conditions under which the community in BIOX reactors could evolve dynamically towards those communities currently observed in commercial operations.

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)).

Effect of sulphate concentration on the community structure and activity of sulphate reducing bacteria

This study investigated the effect of sulphate concentration and residence time on the performance of anaerobic sulphate reduction by a mixed sulphate reducing bacteria (SRB) culture using lactate as the sole carbon source and electron donor. The process performance is related to the population structure of the microbial consortia and dominant metabolic reactions. Laboratory scale chemostat cultures at different residence times (1-4 d) and sulphate concentrations (1.0-10.0 g/L) were employed. Lactate oxidation was prevalent at feed sulphate concentrations of 1.0 to 5.0 g/L. A corresponding increase in the volumetric sulphate reduction rate with increasing volumetric loading rate was also observed at this range. However, at the higher feed sulphate concentration range (10.0-15.0 g/L), sulphate inhibition, lactate fermentation and an increased microbial diversity were evident. At each feed concentration of sulphate in the range 5.0 to 15.0 g/L, varying dilution rates resulted in significant shifts in dominant metabolic reactions. Sulphate concentration and residence time have significant effects on both the structure of the microbial population and kinetics of biological sulphate reduction.

Investigation and Visualisation of Microbial Attachment Trends to Sulphide Minerals in a Bioleach Environment

Two novel experimental approaches have been developed to investigate the attachment of microorganisms to sulphide minerals as a function of the mineral and microbial phase in a joint project with BHP Billiton, conceptualised in 2005 and initiated in 2006. In the first approach, selective attachment of pure cultures to minerals was studied in the “particle coated column reactor” using A. ferrooxidans and L. ferriphilum. The saturated reactor containing glass beads coated with fine mineral concentrates provided a quantifiable surface area of mineral concentrate and maintained good fluid flow. Results are reported for chalcopyrite and pyrite concentrates, a low grade chalcopyrite ore and quartzite. The latter, representing typical gangue material, is used as a control. A. ferrooxidans displayed greater attachment to pyrite, and selective attachment to sulfide minerals over quartz. Similar attachment behaviour trends resulted for Leptospirillum spp. In the second approach, a novel technique was developed to investigate microbial ecology of microbe-mineral attachment, site and mineral specific associations of microorganisms and spatial organisation of microbial communities present. Qualitative assessment and visualisation of microorganisms associated with the mineral surface and subsequent biofilm development was shown in the biofilm reactor, using microscopy techniques and fluorochromes. FISH analyses of A. ferrooxidans and L. ferriphilum on massive chalcopyrite sections are presented. The consequence of the observed attachment on heap bioleach performance is discussed.

Sulfide Mineral Induced Oxidative Stress as a Limiting Factor in Tank Bioleaching Performance

In tank bioleaching, decreasing particle size of milled concentrates has been shown to improve leaching performance owing to increased mineral surface area and mechanical activation effects of fine mineral particles. However, evidence suggests a critical lower limit of particle size distribution exists below which the performance of the thermophilic iron and sulfur oxidizer Sulfolobus metallicus is compromised and complete culture death may result. This paper proposes an explanation for these observations at fine fractions through identifying a relationship between mineral composition of six sulfide concentrates, their extent of milling and the generation of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) in slurries. The effect of oxidative stress induced in the absence of minerals on growth and bioleaching performance of S. metallicus is studied.

Competition between Lactate Oxidisers and Fermenters under Biosulphidogenic Conditions: Implications in the Biological Treatment of AMD

This study investigated the influence of culture conditions on the dominant metabolic pathways and resultant reaction stoichiometry, using chemostat cultures. The kinetic properties (max and Ks) under conditions dominated by either lactate oxidation or lactate fermentation were determined. These properties were used to simulate the competition between lactate fermentation and oxidation. Sulphate reduction and lactate utilisation were determined across residence times of 1 to 5 d and feed sulphate concentrations of 1.0 to 10.0 g l-1. Assuming lactate limitation, results revealed a lower maximal growth rate max of 0.2 h-1 and a higher affinity for lactate characterised by Ks of 0.6 g l-1 for the lactate oxidisers (SRB) than the lactate fermenters. The latter were characterised by a max of 0.3 h-1 and Ks of 3.3 g l-1. Modelling of the competition between lactate fermenters and lactate oxidisers illustrated that lactate oxidisers compete more effectively for lactate under conditions of low lactate concentration (≤5 g l-1) and high sulphide concentration (0.5 g l-1). On the other hand lactate fermenters outcompete the oxidisers under conditions of higher lactate concentration (>5 g l-1). Findings from this study show that in order to optimise BSR system, an understanding of the impact of physicochemical conditions on the metabolic dominance is critical.

The Effect of Nutrient Supplementation on Growth and Leaching Performance of Bioleaching Bacteria

Heap bioleaching operations are often faced with extended and unpredictable lag periods after inoculation, prior to the establishment of a stable oxidising environment, during which the heap is fully colonised or the inoculum overcomes the sub-optimal conditions resulting from acid agglomeration. Supplementation of laboratory scale (4kg ore) leach columns with soluble nitrogen, particularly as yeast extract, significantly reduced the lag time and promoted bacterial growth, resulting in a 50-95% increase in copper recovery post-inoculation. The effect of yeast extract addition to Acidithiobacillus ferrooxidans in controlled oxidation tests was investigated. Initial exposure of a stock culture to yeast extract resulted in a transient, dose dependent inhibition at concentrations of 0.5 g.l-1 and below. At 1.25 g.l-1 inhibition was complete over the time scale of the experiment. The inhibition phase was characterised by observable changes in cell morphology and ultrastructure. Despite the initial inhibition, the biomass yield at the end of the experiments was equivalent, or higher, in the presence of yeast extract. Cultures were adapted to growth on yeast extract as the sole nitrogen source and adapted cultures showed the highest rates of iron oxidation and cell growth, in the presence of 0.5 and 1 g.l-1 of yeast extract.