A biomining approach to process low-grade iron ores has the potential to turn closed mines or uneconomic mineral deposits into economic resources. Microorganisms and their metabolites have been commercially applied in the leaching of metals from medium- and low-grade sulfide minerals for many years. Efforts are now being directed to the application of biomining to oxide ore systems as high-grade ore becomes scarce. This chapter discusses the potential exploitation of microorganisms and their metabolites for the bioleaching of phosphorus from iron ore and as bioreagents for the selective flotation and flocculation of iron ore minerals.

The efficiency of chalcopyrite bioleaching in a high sulfate background was evaluated using acidophilic microorganisms adapted to sulfate. The concentration of magnesium sulfate added to mesophilic, moderately thermophilic and thermophilic bioleaching tests was equivalent to 100, 40 and 80 g L-1SO42-, respectively. Biological copper extraction was highest at 45 °C (67 %), followed by 60 °C (54 %) and 30 °C (16 %). Quantitative x-ray diffraction (QXRD) analysis of the ROM ore and bioleached residues revealed the complete disappearance of pyrrhotite and a significant reduction of pyrite at all temperatures. Significant chalcopyrite was leached at 45 and 60 °C; however, no chalcopyrite was leached at 30 °C. As the bioleach did not plateau after 31 days, it is possible that higher copper yields may have been achieved with prolonged leaching.

Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P > 0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.

As the quality of gold (Au) deposits decreases, processes which can economically extract Au from low grade ores will grow in importance to the minerals industry. In situ, in-place and vat leaching methods can be attractive alternatives for low grade ores that are uneconomical to process using conventional open-pit or underground mining and processing methods. The objective of this study was to evaluate at laboratory scale a submerged bio-oxidation process concept to potentially improve the recovery of Au from refractory ore bodies that may be amenable to in-place or vat processing. The process included biological generation of ferric iron in a bioreactor followed by ferric iron oxidation of pyrite in submerged upflow columns. The results showed that oxidation of pyrite is possible using ferric iron that is biologically generated either externally or using underground aeration in the ore body. The simulated underground aeration and the presence of bioleaching microorganisms clearly enhanced the oxidation of pyrite. Moreover, microorganisms decreased the accumulation of elemental-S, the presence of which may have a negative effect on subsequent gold leaching and recovery.

ABSTRACT The objectives of this study were to formulate a vaccine based upon the different species/strains of methanogens present in sheep intended to be immunized and to determine if a targeted vaccine could be used to decrease the methane output of the sheep. Two 16S rRNA gene libraries were used to survey the methanogenic archaea in sheep prior to vaccination, and methanogens representing five phylotypes were found to account for >52% of the different species/strains of methanogens detected. A vaccine based on a mixture of these five methanogens was then formulated, and 32 sheep were vaccinated on days 0, 28, and 103 with either a control or the anti-methanogen vaccine. Enzyme-linked immunosorbent assay analysis revealed that each vaccination with the anti-methanogen formulation resulted in higher specific immunoglobulin G titers in plasma, saliva, and rumen fluid. Methane output levels corrected for dry-matter intake for the control and treatment groups were not significantly different, and real-time PCR data also indicated that methanogen numbers were not significantly different for the two groups after the second vaccination. However, clone library data indicated that methanogen diversity was significantly greater in sheep receiving the anti-methanogen vaccine and that the vaccine may have altered the composition of the methanogen population. A correlation between 16S rRNA gene sequence relatedness and cross-reactivity for the methanogens ( R 2 = 0.90) also exists, which suggests that a highly specific vaccine can be made to target specific strains of methanogens and that a more broad-spectrum approach is needed for success in the rumen. Our data also suggest that methanogens take longer than 4 weeks to adapt to dietary changes and call into question the validity of experimental results based upon a 2- to 4-week acclimatization period normally observed for bacteria.

Four formate-utilizing methanogens were isolated from ovine (strain KM1H5-1P(T)) and bovine (strains AK-87, OCP and ZA-10(T)) rumen contents. Based on 16S rRNA gene sequence analysis, the methanogen strains were found to belong to the order Methanobacteriales in the genus Methanobrevibacter. Strains ZA-10(T) and KM1H5-1P(T) gained energy for growth by the reduction of CO(2) to CH(4) using H(2) or formate exclusively as electron donors. Increasing formate concentrations to 220 mM in batch cultures increased the growth of strain KM1H5-1P(T) but did not affect the growth of strain ZA-10(T). Substrate specificity and resistance to cell-wall lysis supported the affiliation of the strains to the genus Methanobrevibacter. Strains ZA-10(T) and KM1H5-1P(T) showed 16S rRNA gene sequence similarity of 98.0 and 98.6 % to their closest recognized relatives, Methanobrevibacter thaueri CW(T) and Methanobrevibacter ruminantium M1(T), respectively. DNA-DNA hybridization experiments indicated that the strains were not affiliated at the species level to their closest recognized relatives, with DNA reassociation values of only 28 % between strains ZA-10(T) and Methanobrevibacter thaueri CW(T) and <25 % between strains KM1H5-1P(T) and Methanobrevibacter ruminantium M1(T). Based on the data presented, the new strains are considered to represent two novel species of the genus Methanobrevibacter, for which the names Methanobrevibacter millerae sp. nov. (type strain ZA-10(T)=DSM 16643(T)=OCM 820(T)) and Methanobrevibacter olleyae sp. nov. (type strain KM1H5-1P(T)=DSM 16632(T)=OCM 841(T)) are proposed.

Two rumen protozoa vaccine formulations containing either whole fixed Entodinium or mixed rumen protozoa cells were tested on Merino sheep with the aim of decreasing the number and/or activity of protozoa in the rumen. Negative control (no antigen) and positive control (Tetrahymena corlissi antigens) treatments were also included in the experiment. Blood and saliva were sampled to measure the specific immune response. Protozoal numbers in the rumen were monitored by microscopic counts. Vaccination with protozoal formulations resulted in the presence of specific IgG in plasma and saliva, but saliva titres were low. Titres after secondary vaccination were higher (P < 0·05) than after primary vaccination. There was a moderate (r2 0·556) relationship (P < 0·05) between plasma and saliva titres for the rumen protozoal vaccine formulations. Rumen protozoa were not decreased (P>0·05) by the vaccination and there was also no difference (P>0·05) between treatments in rumen fluid ammonia-N concentration or wool growth. In vitro studies investigated the binding ability of the antibodies and estimated the amount of antibody required to reduce cell numbers in the rumen. The studies showed that the antibodies did bind to and reduced protozoa numbers, but the amount of antibody generated by vaccination was not enough to produce results in an in vivo system. It is suggested that the vaccine could be improved if specific protozoal antigens are determined and isolated and that improved understanding of the actions of protozoa antibodies in rumen fluid and the relationships between levels of antibodies and numbers of protozoa in the rumen is needed.

This work was conducted to determine if methane emissions from sheep immunized with an anti-methanogen vaccine were significantly lower than methane emissions from non-immunized sheep, to test the effectiveness of two different vaccine formulations (VF) on methane abatement, and to compare methane emissions measured using a closed-circuit respiration chamber and the sulphur-hexafluoride (SF6) tracer technique. Thirty mature wether sheep were randomly allocated to three treatment groups (n = 10). One group received an immunization of adjuvant only on days 0 and 153 (control), a second group received an immunization with a 3-methanogen mix on days 0 and 153 (VF3 + 3), and a third group received an immunization of a 7-methanogen mix on day 0 followed by a 3-methanogen mix on day 153 (VF7 + 3). Four weeks post-secondary immunization, there was a significant 7.7% reduction in methane production per kg dry matter intake in the VF7 + 3 group compared to the controls (P = 0.051). However, methane emissions from sheep immunized with VF7 + 3 were not significantly different when compared to the sheep in the control group (P = 0.883). The average IgG and IgA antibody titres in both plasma and saliva of the VF3 + 3 immunized sheep were four to nine times higher than those immunized with VF7 + 3 (P< 0.001) at both 3 and 6 weeks post-secondary immunization. Data also revealed that SF6 methane estimates were consistently higher than the respiration chamber estimates and that there was no significant correlation between the SF6 methane estimates and the respiration chamber methane estimates (R2 = 0.11).

16S rRNA clone library analysis was used to examine the biodiversity and community structure within the sediments of three hypersaline Antarctic lakes. Compared to sediment of low to moderate salinity Antarctic lakes the species richness of the hypersaline lake sediments was 2–20 times lower. The community of Deep Lake (32% salinity, average sediment temperature −15°C) was made up almost entirely of halophilic Archaea. The sediment communities of two meromictic hypersaline lakes, Organic Lake (20% salinity, −7°C) and Ekho Lake (15% salinity, 15°C) were more complex, containing phylotypes clustering within the Proteobacteria and Cytophagales divisions and with algal chloroplasts. Many phylotypes of these lakes were related to taxa more adapted to marine-like salinity and perhaps derive from bacteria exported into the sediment from the lower salinity surface waters. The Ekho Lake clone library contained several major phylotypes related to the Haloanaerobiales, the growth of which appears to be promoted by the comparatively high in situ temperature of this lake.

16S rDNA clone library analysis was used to examine the biodiversity and community structure within anoxic sediments of several marine-type salinity meromictic lakes and a coastal marine basin located in the Vestfolds Hills area of Eastern Antarctica. From 69 to 130 (555 total) 16S rDNA clones were analysed from each sediment sample, and restriction fragment length polymorphism (RFLP) and sequence analysis grouped the clones into 202 distinct phylotypes (a clone group with sequence similarity of >0.98). A number of phylotypes and phylotype groups predominated in all libraries, with a group of 10 phylotypes (31% of clones) forming a novel deep branch within the low G+C Gram-positive division. Other abundant phylotypes detected in several different clone libraries grouped with Prochlorococcus cyanobacteria, diatom chloroplasts, delta proteobacteria (Desulfosarcina group, Syntrophus and Geobacterl Pelobacter/Desulphuromonas group), order Chlamydiales (Parachlamydiaceae) and Spirochaetales (wall-less Antarctic spirochaetes). Most archaeal clones detected (3.1% of clones) belonged to a highly diverged group of Euryarchaeota clustering with clones previously detected in rice soil, aquifer sediments and hydrothermal vent material. Little similarity existed between the phylotypes detected in this study and other clone libraries based on marine sediment, suggesting that an enormous prokaryotic diversity occurs within marine and marine-derived sediments.

A polyphasic taxonomic study was performed to characterize dissimilatory iron-reducing strains mostly isolated from Antarctic sea ice. The strains were isolated from samples of congelated (land-fast) sea ice, grease ice, and ice algal biomass collected from the coastal areas of the Vestfold Hills in eastern Antarctica (68 degrees S 78 degrees E). The strains were facultatively anaerobic, motile, and rod shaped, were capable of anaerobic growth either by fermentation of carbohydrates or by anaerobic respiration, and utilized a variety of electron acceptors, including nitrate, ferric compounds, and trimethylamine N-oxide. A phylogenetic analysis performed with 16S rRNA sequences showed that the isolates formed two groups representing novel lineages in the genus Shewanella. The first novel group included seawater-requiring, psychrophilic, chitinolytic strains which had DNA G + C contents of 48 mol%. The members of the second strain group were psychrotrophic and did not require seawater but could tolerate up to 9% NaCl. The strains of this group were also unable to degrade polysaccharides but could utilize a number of monosaccharides and disaccharides and had G + C contents of 40 to 43 mol%. The whole-cell-derived fatty acid profiles of the sea ice isolates were found to be similar to the profiles obtained for other Shewanella species. The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) (20:5 omega 3) was detected in all of the sea ice isolates at levels ranging from 2 to 16% of the total fatty acids. EPA was also found at high levels in Shewanella hanedai (19 to 22%) and Shewanella benthica (16 to 18%) but was absent in Shewanella alga and Shewanella putrefaciens. On the basis of polyphasic taxonomic data, the Antarctic iron-reducing strains are placed in two new species, Shewanella frigidimarina sp. nov. (type strain, ACAM 591) and Shewanella gelidimarina sp. nov. (type strain, ACAM 456).