A. G. Bulaev

Complete nitrification by Nitrospira bacteria

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle

Nitrification, the oxidation of ammonia (NH3) via nitrite (NO2−) to nitrate (NO3−), is a key process of the biogeochemical nitrogen cycle. For decades, ammonia and nitrite oxidation were thought to be separately catalysed by ammonia-oxidizing bacteria (AOB) and archaea (AOA), and by nitrite-oxidizing bacteria (NOB). The recent discovery of complete ammonia oxidizers (comammox) in the NOB genus Nitrospira, which alone convert ammonia to nitrate, raised questions about the ecological niches in which comammox Nitrospira successfully compete with canonical nitrifiers. Here we isolate a pure culture of a comammox bacterium, Nitrospira inopinata, and show that it is adapted to slow growth in oligotrophic and dynamic habitats on the basis of a high affinity for ammonia, low maximum rate of ammonia oxidation, high growth yield compared to canonical nitrifiers, and genomic potential for alternative metabolisms. The nitrification kinetics of four AOA from soil and hot springs were determined for comparison. Their surprisingly poor substrate affinities and lower growth yields reveal that, in contrast to earlier assumptions, AOA are not necessarily the most competitive ammonia oxidizers present in strongly oligotrophic environments and that N. inopinata has the highest substrate affinity of all analysed ammonia oxidizer isolates except the marine AOA Nitrosopumilus maritimus SCM1 (ref. 3). These results suggest a role for comammox organisms in nitrification under oligotrophic and dynamic conditions.

Bioprocessing of Mining and Metallurgical Wastes Containing Non-Ferrous and Precious Metals

Mining and metallurgical treatments of sulphide ores are characterised by present significant losses of non-ferrous and precious metals as different types of waste. These elements are accumulated in heaps due to the lack of efficient technology for the recovery of the metals from metallurgical waste. The treatment of two types of industrial metallurgical waste (copper converter slag and old flotation pyrite tailings) containing non-ferrous and precious metals were examined in the laboratory. Leaching of the slag containing 2.74% Cu (as digenite, bornite, and free metal) and 2.49% Zn (as a ferrite ZnFe2O4 and silicate) by an Fe3+-containing solution was studied. The effect of various experimental parameters on the leaching dynamics of copper, zinc, and iron under batch conditions was investigated. The following experimental parameters were recommended: a pH of 1.5, a pulp density of 10% (w/v), a temperature of 70 °C, and an initial Fe3+ concentration of 15 g/L. Leaching under these conditions resulted in the solubilisation of 89.4% copper and 35.3% zinc within 2.5 hours. Percolation leaching of the pyrite tailings containing 0.29% Cu (as chalcopyrite), 0.26% Zn (as sphalerite), 0.00007% gold, and 0.00108% silver was also studied. Acidic percolation leaching and the resulting biooxidation lasting 134 days resulted in the solubilisation of 73.4% zinc and 50.8% copper. The recovery rates of gold and silver from the bioleaching residues by cyanidation were 57.2% and 50.7%, respectively. The data obtained in the present work may be used to estimate the operating parameters for the industrial-scale processing of non-ferrous and precious metals from mining and metallurgical waste.

Leaching of rare earth elements from coal ashes using acidophilic chemolithotrophic microbial communities

A method for leaching rare earth elements from coal ash in the presence of elemental sulfur-oxidizing communities of acidophilic chemolithotrophic microorganisms was proposed. The optimal parameters determined for rare element leaching in reactors were as follows: temperature, 45°C; initial pH, 2.0; pulp density, 10%; and the coal ash to elemental sulfur ratio, 10 : 1. After ten days of leaching, 52.0, 52.6, and 59.5% of scandium, yttrium, and lanthanum, respectively, were recovered.

Biooxidation of a gold-containing sulfide concentrate in relation to changes in physical and chemical conditions

The growth of a microbial community and the oxidation of iron- and sulfur-containing substrates in batch culture during the leaching/oxidation of the flotation concentrate of refractory gold-arsenic sulfide ore were optimized with respect to the following medium parameters: temperature, pH, and requirement in organic substances. It was revealed that the optimum mode is (i) to maintain the pH at 1.6–1.7 and the temperature at 34–35 and 38°C and (ii) to add Corg in the form of yeast extract (0.02%). Mutually beneficial or competitive relationships among groups of microorganisms of the community were established, depending on the cultivation conditions.

Changes in the species composition of a thermotolerant community of acidophilic chemolithotrophic microorganisms upon switching to the oxidation of a new energy substrate

Construction and analysis of the 16S rDNA clone libraries was used to investigate the species composition of two thermotolerant communities of acidophilic chemolithotrophic microorganisms (ACM) isolated from the pulp of laboratory reactors used for oxidation of different gold-containing ore concentrates. The first community was formed during oxidation of the pyrite-arsenopyrite ore concentrate from the Kyuchus deposit. The clones of the bacterial component of this community belonged to the genera Sulfobacillus (32 clones) and Leptospirillum (33 clones). The Sulfobacillus clones belonged to three groups: Sb. thermosulfidooxidans, Sb. benefaciens, and Sb. thermotolerans. All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. The microorganisms of this community were used as inoculum for biooxidation of a different mineral concentrate, the pyrrhotite-containing pyrite-arsenopyrite ore concentrate from the Olympiadinskoe deposit, and the structure of the community formed in the process was investigated. The clones of the bacterial component of the second community also belonged to the genera Sulfobacillus (14 clones) and Leptospirillum (48 clones). The Sulfobacillus clones belonged to the species Sb. thermosulfidooxidans (13 clones) and Sb. thermotolerans (1 clone). All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. During the adaptation of the community to a new oxidized mineral substrate, both the composition and the ratio of the constituent microbial species changed.

Growth of acidophilic chemolithotrophic microbial communities and sulfur oxidation in the presence of coal ashes

Out of two communities of acidophilic chemolithotrophic microorganisms (ACM), a mesophilic and a thermophilic ones, the latter was selected due to its higher rate of sulfur oxidation and pH decrease to >1 in the presence of coal ashes (CA). The structure of ACM communities was retained. Sulfur-oxidizing activity of the microorganisms from the thermophilic community was observed at pH from 1.8 to 2.6 at sulfur concentrations of 0.6–1.0%. Efficient production of acidic solutions with pH 0.69–0.86 was observed after single CA introduction in the amount corresponding to 5–30% pulp density and in the course of pulp density increasing daily from 1 to 50%. Leaching of rare earth elements from 30% coal ash pulp (pH 0.76) was 15 to 30% of their initial content. No sorption of metals on the biomass was observed.

Selection of a community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite-containing sulphide ore flotation concentrate

A community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite-containing sulphide ore flotation concentrate was selected. The Acidithiobacillus caldus OP-1 and Ferroplasma acidiphilum OP-2 cultures were identified to be dominating members. The presence of the Acidithiobacillus ferrooxidans OP-3, Leptospirillum ferriphilum OP-4, and Sulfobacillus thermosulfidooxidans OP-5 cultures in the community’s composition was also mentioned. The analysis results of solid residues of the process showed a greater elemental sulfur oxidation level and gold recovery when the initial pH value in tank I was maintained at a level of 1.8–2.0 (90.5%) rather than 1.6–1.8 (86.3%).

Biooxidation of a double-refractory gold-bearing sulfide ore concentrate

The efficiency of biooxidation for treatment of a double-refractory gold-bearing sulfide ore concentrate from the Bakyrchik deposit (East Kazakhstan) was defined. The experiments were conducted in two different modes, i.e., with the standard liquid medium and the medium imitating the chemical composition of the Bakyrchik deposit groundwater and containing high concentrations of sodium, magnesium, and chloride. The concentrate contained 17.5% of organic carbon, 6% of pyrite, and 13% of arsenopyrite. Gold content was 57.5 g t–1. Direct gold recovery by cyanidation was very low (2.8%). While biooxidation was efficient in both cases (approximately 90% of sulfide sulfur was oxidized), the efficiency of cyanidation was low (39 and 32%, respectively). This fact suggests high efficiency of biooxidation is insufficient for efficient treatment of double-refractory gold-bearing sulfide ore concentrates.

Effect of the aeration mode and yeast extract on the oxidation of high-pyrrhotite sulfide ore flotation concentrate and on the composition of the acidophilic chemolithotrophic microbial community

Optimal aeration conditions were determined and the effect of yeast extract on biooxidation of high-pyrrhotite sulfide ore flotation concentrate in the course of continuous cultivation of an acidophilic chemolithotrophic microbial community was studied in a line of four sequential laboratory reactors; the aeration rate was 3 L/(L min), yeast extract concentration was 0.02%. The gold recovery level was 96.45% at 2.23% elemental sulfur content in the solid residue. The dominant strains identified in the community responsible for biooxidation were Acidithiobacillus caldus OL13-1, At. caldus OL13-3 = At. caldus OL12-3, and an ‘Acidiferrobacter’ strain. Strains Sulfobacillus thermosulfidooxidans OL13-2 = S. thermosulfidooxidans OL12-1 and Ferroplasma acidiphilum OL13-4 = F. acidiphilum OL12-4 were isolated in pure culture and identified.