Bioleaching of chalcopyrite concentrate by a moderately thermophilic culture in a stirred tank reactor

A mixed culture of moderately thermophilic microorganisms was enriched from acid mine drainage samples collected from several chalcopyrite mines in China. Such mixed culture can be used to effectively extract copper from chalcopyrite. Furthermore, after being adapted to gradually increased concentration of chalcopyrite concentrate, the tolerance of the mixed culture to chalcopyrite concentrate was brought up to 80 g/L. The effects of several leaching parameters on copper recovery in stirred tank reactor also had been investigated. The results of the investigation show that it was possible to achieve a copper extraction rate of 75% in 44 days at a pulp density of 8%. The leaching rate of chalcopyrite concentrate tended to increase with dissolved total iron concentration. At low pH ranges, more microscopic counts of microorganisms were found in the solution. Furthermore, the analysis of leached residues indicates that the passivation of chalcopyrite concentrate was mainly due to a mass of jarosite and PbSO(4) on the mineral surface, other than the elemental sulphur layer. The bacterial community composition was analyzed by using Amplified Ribosomal DNA Restriction Analysis. Two moderately thermophilic bacteria species were identified as Leptospirillum ferriphilum and Acidithiobacillus caldus with abundance of 67% and 33% in the bio-pulp, respectively.     

Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost

Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/mol-hexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the' PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticium was isolated in the enrichment culture with or without added peptone. and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from cellulose by microflora is performed by a consortium of several species of microorganisms.     

光照混菌培养应用研究进展

光合细菌与其他微生物在光照条件下混合培养是近年来的研究热点。综述了光照混菌培养的特点和目前光照混菌培养在水体净化、生物制氢和高价值物质生产方面的应用,并对影响混合菌株生长代谢与繁殖的因素做了总结。分析表明菌株之间存在的相互协同共生作用能促进微生物的生长繁殖,使底物被充分利用,提高物质产率。光照混菌培养工艺简单、成本较低,在水体净化、生物制氢、高价值物质生产方面的应用具有相当好的效果。在影响因素中对混合培养影响最大的因素是菌株接种量、接种比和培养基pH。在总结光照混菌培养应用现存不足的基础上,对其发展前景作出展望。     

Two-stage process of bacterial-chemical oxidation of refractory pyrite-arsenopyrite gold-bearing concentrate

A technology for tank biooxidation of refractory gold-bearing concentrate under variable temperature conditions has been improved: the temperature of the first of two stages was changed from 30°C to 34–36°C. Gold in this concentrate is mainly associated with sulfide minerals: arsenopyrite and pyrite, which underlies a low gold recovery (16.68%) as a result of cyanidation. To resolve the problem, an association of mesophilic acidophilic chemolithotrophic microorganisms and moderately thermophilic bacteria of the Sulfobacillus genus were used for the concentrate oxidation. The composition of the used microbial association was studied; it was shown that it depends upon temperature: at 42°C, the population of the mesophilic thiobacteria decreased, whereas that of thermophilic sulfobacilli enhanced as compared to 36°C. The accepted scheme of the process ensures a high extent of gold recovery (94.6%) within a short space of time for biooxidation (96 h).     

Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts

Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case individually.     

高温油藏内源微生物及其提高采收率潜力研究

大港孔店油田油藏特征、流体和微生物性质分析结果表明, 属于高温生态环境, 地层水矿化度较低, 氮、磷浓度低, 而且缺乏电子受体, 主要的有机物来源是油气。油田采用经过除油处理的油藏产出水回注方式开发, 油层中存在的微生物类型主要是厌氧嗜热菌, 包括发酵菌(102个/mL~105个/mL), 产甲烷菌(103个/mL); 好氧菌主要存在于注水井周围。硫酸盐还原菌(SRB)还原速率0.002 mg S2-/(L·d) ~18.9 mg S2-/(L·d), 产甲烷菌产甲烷速率0.012 mgCH4/(L·d)~16.2 mgCH4/(L·d)。好氧菌能够氧化油形成生物质, 部分氧化产物为挥发性脂肪酸和表面活性剂。产甲烷菌在油氧化菌液体培养基中产生CH4, CO2为好氧微生物和厌氧微生物的共同代谢产物。这些产物具有提高原油流动性的作用。用示踪剂研究了注入水渗流方向。通过综合分析, 油藏微生物具有较大的潜力, 基于激活油层菌的提高采收率方法在该油田是可行的。     

高温油藏内源微生物及其提高采收率潜力研究

大港孔店油田油藏特征、流体和微生物性质分析结果表明,属于高温生态环境,地层水矿化度较低,氮、磷浓度低,而且缺乏电子受体,主要的有机物来源是油气.油田采用经过除油处理的油藏产出水回注方式开发,油层中存在的微生物类型主要是厌氧嗜热菌,包括发酵菌(102个/mL～105个/mL),产甲烷菌(103个/mL);好氧菌主要存在于注水井周围.硫酸盐还原菌(SRB)还原速率0.002 μg S2-/(L·d)～18.9 μg S2-/(L·d),产甲烷菌产甲烷速率0.012 μgCH4/(L·d)～16.2 μgCH4/(L·d).好氧菌能够氧化油形成生物质,部分氧化产物为挥发性脂肪酸和表面活性荆.产甲烷菌在油氧化菌液体培养基中产生CH4,CO2为好氧微生物和厌氧微生物的共同代谢产物.这些产物具有提高原油流动性的作用.用示踪剂研究了注入水渗流方向.通过综合分析,油藏微生物具有较大的潜力,基于激活油层茵的提高采收率方法在该油田是可行的.     

High-level expression of extracellular secretion of a β-xylosidase gene from Paecilomyces thermophila in Escherichia coli

A novel β-xylosidase gene (designated as PtXyl43) from thermophilic fungus Paecilomycesthermophila was cloned and extracellularly expressed in Escherichia coli. PtXyl43 belonging to glycoside hydrolase (GH) family 43 has an open reading frame of 1017 bp, encoding 338 amino acids without a predicted signal peptide. No introns were found by comparison of the PtXyl43 genomic DNA and cDNA sequences. The recombinant β-xylosidase (PtXyl43) was secreted into the culture medium in E. coli with a yield of 98.0 U mL(-1) in shake-flask cultures. PtXyl43 was purified 1.2-fold to homogeneity with a recovery yield of 61.5% from the cell-free culture supernatant. It appeared as a single protein band on SDS-PAGE with a molecular mass of approx 52.3 kDa. The enzyme exhibited an optimal activity at 55 °C and pH 7.0, respectively. This is the first report on the cloning and expression of a GH family 43 β-xylosidase gene from thermophilic fungi.     

Detection of a Culturable Hyperthermophilic Archaeon of the Genus Sulfophobococcus in an Anaerobic Digestor Operated in a Thermophilic Regime

A stable association of hyperthermophilic microorganisms (82°C), which contained mostly cocci and a minor amount of non-spore-forming rods, was obtained from the digested sludge of an anaerobic digestor used to process municipal wastewater under thermophilic conditions (50°C). PCR amplification of 16S rRNA genes using total DNA isolated from this association and archaea-specific primers, followed by sequencing of the product obtained, showed that the archaeal component was represented by a single nucleotide sequence, which was 99.9% homologous to the 16S rRNA gene of Sulfophobococcus zilligii. Thus, a hyperthermophilic archaeon was for the first time detected in a system of anaerobic biological treatment of wastewater. In addition, this is the first report on the detection of a culturable member of Crenarchaeota in anthropogenic habitats with neutral pH.     

Engineering of microorganisms towards recovery of rare metal ions

The bioadsorption of metal ions using microorganisms is an attractive technology for the recovery of rare metal ions as well as removal of toxic heavy metal ions from aqueous solution. In initial attempts, microorganisms with the ability to accumulate metal ions were isolated from nature and intracellular accumulation was enhanced by the overproduction of metal-binding proteins in the cytoplasm. As an alternative, the cell surface design of microorganisms by cell surface engineering is an emerging strategy for bioadsorption and recovery of metal ions. Cell surface engineering was firstly applied to the construction of a bioadsorbent to adsorb heavy metal ions for bioremediation. Cell surface adsorption of metal ions is rapid and reversible. Therefore, adsorbed metal ions can be easily recovered without cell breakage, and the bioadsorbent can be reused or regenerated. These advantages are suitable for the recovery of rare metal ions. Actually, the cell surface display of a molybdate-binding protein on yeast led to the enhanced adsorption of molybdate, one of the rare metal ions. An additional advantage is that the cell surface display system allows high-throughput screening of protein/peptide libraries owing to the direct evaluation of the displayed protein/peptide without purification and concentration. Therefore, the creation of novel metal-binding protein/peptide and engineering of microorganisms towards the recovery of rare metal ions could be simultaneously achieved.     

The application of triazine dye affinity chromatography to the large-scale purification of glycerokinase from Bacillus stearothermophilus

Gram quantities of homogeneous glycerokinase have been prepared from the thermophilic bacterium, Bacillus stearothermophilus, using three major steps: precipitation of debris at pH 5.1, ion-exchange chromatography on DEAE-Sephadex, and affinity chromatography on Procion Blue MX-3G-Sepharose. This method is a considerable improvement over conventional techniques; the purified enzyme was obtained with a 40% recovery and a specific activity of 120 units (mumol/min)/mg protein. A modified culture medium enabled yields of 3.4 X 10(6) units of enzyme to be obtained from 400-liter production cultures.     

Thermophilic vs. mesophilic bioleaching process performance

Abstract: Mesophilic and thermophilic mineral oxidising microbial cultures were compared for their capacity to leach both a complex pyritic and an arsenopyritic ore, aiming at copper and gold recovery, respectively. The mesophilic cultures are primarily based on the activity of Thiobacillus -type microorganisms, while the thermophile characteristics place them in the sulphur metabolizers branch df Archaebacteria. Study of key process variables and determination of their optimum values were carried out to provide a basis for significant physiological and technical comparison. Each microbial culture finds a preferential process application, depending on the choice of the reactor and ore.     

Immobilised activated sludge based biosensor for biochemical oxygen demand measurement

A biochemical oxygen demand (BOD) sensor, based on an immobilised mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4–8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD₅ analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below ±5.6% standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz. ±15% standard deviation.     

Thermophilic aerobic treatment of a synthetic wastewater in a membrane-coupled bioreactor

Synthetic wastewater containing -lactose and gelatin was treated in a thermophilic membrane-coupled bioreactor (MBR). Thermophilic (>45°C) treatment represents a potentially advantageous process for high-temperature as well as high-strength industrial wastewaters susceptible to reactor autoheating. Thermophilic systems, however, generally support a nonflocculating biomass that resists conventional methods of cell separation from the treated wastewater. MBRs were applied to thermophilic treatment systems because bacterial cells can be retained regardless of cell aggregation. Thermophilic aerobic MBRs were successfully operated at high levels of biocatalyst and produced a better effluent quality than analogous thermophilic bioreactors without cell recycle. At a hydraulic residence time (HRT) of 13.1 h, the chemical oxygen demand (COD) of the membrane eluate improved from 760 mg l⁻¹ (without cell recycle) to 160 mg l⁻¹ (with cell recycle). Bacterial community shifts were detected by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) -amplified 16S rRNA gene fragments — 6 of 13 bands disappeared within 2 days of MBR operation. A concomitant 40–50% reduction in physiological indicators of cell reactivity (RNA:protein; ATP:protein) was also observed. The specific activity of β-galactosidase and aminopeptidase, however, increased by 10–25%, indicating that there is a definite advantage to MBR operation at the highest biomass level possible. Nucleotide sequence analysis of 16S rDNA clones identified phylotypes from the low-G+C Gram-positive division and the β- and γ-subdivisions of Proteobacteria. Journal of Industrial Microbiology & Biotechnology (2001) 26, 203–209. Received 18 March 2000/ Accepted in revised form 26 January 2001     

Thermophilic Microbial Communities of Deep-Sea Hydrothermal Vents

The most recent publications on the phylogenetic and functional diversity of thermophilic prokaryotes inhabiting thermal deep-sea environments are reviewed. Along with a general physicochemical characterization of the biotope studied, certain adaptation mechanisms are discussed that are peculiar to the microorganisms inhabiting it. A separate chapter addresses the phylogenetic analysis of deep-sea hydrothermal microbial communities and uncultivated microorganisms recently discovered therein using molecular biological techniques. Physiological groups of thermophilic microorganisms found in deep-sea hydrothermal vents are considered: methanogens, sulfate-, iron-, and sulfur-reducers, aerobic hydrogen-oxidizing prokaryotes, aerobic and anaerobic organotrophs. In most cases, the isolates represent novel taxons.     

Degradation of cyanide by a bacterial mixture composed of new types of cyanide-degrading bacteria

Summary A mixed culture of bacteria capable of growth on cyanide was isolated from an activated sludge of coal tar wastewater by an enrichment culture technique. The predominant cyanide-degrading microorganisms found in this bacterial mixture were identified as species of the genera Klebsiella, Serratia, Moraxella, and Pseudomonas. Stoichiometric amounts of ammonia were released during the cyanide containing culture by microbial oxidation of cyanide.     

Microbiological investigations of high-temperature horizons of the Kongdian petroleum reservoir in connection with field trial of a biotechnology for enhancement of oil recovery

The physicochemical conditions and microbiological characteristics of the formation waters of the Kongdian oilfield of the Dagang oilfield (China) were studied. It was demonstrated that this oilfield is a high-temperature ecosystem with formation waters characterized by low mineralization. The concentrations of nitrogen and phosphorus compounds, as well as of electron acceptors, are low. Oil and oil gas are the main organic matter sources. The oilfield is exploited with water-flooding. The oil stratum was inhabited mostly by anaerobic thermophilic microorganisms, including fermentative (10²–10⁵ cells/ml), sulfate-reducing (0–10² cells/ml), and methanogenic (0–10³ cells/ml) microorganisms. Aerobic bacteria were detected mainly in the near-bottom zone of injection wells. The rate of sulfate reduction varied from 0.002 to 18.940 μg S^2? l^?1 day^?1 and the rate of methanogenesis from 0.012 to 16.235 μg CH₄ l^?1 day^?1. Microorganisms with great biotechnological potential inhabited the oilfield. Aerobic thermophilic bacteria were capable of oxidizing oil with formation of biomass, the products of partial oxidation of oil (volatile acids), and surfactants. During growth on the culture liquid of oil-oxidizing bacteria, methanogenic communities produced methane and carbon dioxide, which also had oil-releasing capabilities. Using various labeled tracers, the primary filtration flows of injected solutions at the test site were studied. Our comprehensive investigations allowed us to conclude that the method for microbial enhancement of oil recovery based on the activation of the stratal microflora can be applied in the Kongdian oilfield.     

Thermophilic microbial communities of deep-sea hydrothermal environments

The latest publications on the phylogenetic and functional diversity of thermophilic prokaryotes inhabiting thermal deep-sea environments are reviewed. Along with general physicochemical characterization of the biotope studied, certain adaptation mechanisms are discussed that are peculiar to the microorganisms inhabiting it. A separate chapter addresses phylogenetic analysis of deep-sea hydrothermal microbial communities and uncultivated microorganisms recently discovered therein using molecular biological techniques. Physiological groups of thermophilic microorganisms found in deep-sea hydrotherms are considered: methanogens, sulfate-, iron-, and sulfur-reducers, aerobic hydrogen-oxidizing prokaryotes, aerobic and anaerobic organotrophs. In most cases, the isolates represent novel taxa.     

Isolation and characterization of a filamentous viruslike particle from Clostridium acetobutylicum NCIB 6444.

A single-stranded 6.6-kb DNA molecule complexed with protein was recovered from the supernatant of Clostridium acetobutylicum NCIB 6444. Electron microscopic examination of the DNA-protein complex revealed the presence of a filamentous viruslike particle, which was designated CAK1. The possible double-stranded plasmidlike replicative form and the single-stranded prophage were also recovered from the cell culture following alkaline lysis. CAK1 was released from the C. acetobutylicum cell culture in the absence of cell lysis. Polyethylene glycol-NaCl coprecipitation of the DNA-protein complex revealed the presence of single-stranded DNA complexed with protein in a manner rendering the DNA resistant to Bal 31 exonuclease. Proteinase treatment of CsCl density gradient-purified CAK1 resulted in recovery of DNase-sensitive single-stranded DNA. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CAK1 demonstrated the presence of a 5-kDa major coat protein. Hybridization data indicated that the single-stranded DNA from CAK1 has homology with the M13 phage of Escherichia coli. An examination of various physical properties of CAK1 suggests that it is similar to the filamentous phage recovered from gram-negative microorganisms. Although infectivity or inducibility of CAK1 could not be demonstrated, to our knowledge this represents the first report of a nonlytic filamentous viruslike particle containing single-stranded DNA being recovered from a gram-positive bacterium.     

Selective leaching of zinc from copper-zinc concentrate

Biooxidation of copper-zinc concentrate with the use of consortia of mesophilic and moderately thermophilic acidophilic chemolithotrophic microorganisms was studied. Pyrrhotite, sphalerite, and chalcopyrite were the main sulfide minerals of the concentrate. The possibility in principal of complete selective leaching of zinc from sulfide concentrate coupled with minimal recovery of copper (less than 20%) was demonstrated. Selective leaching of zinc could be caused by galvanic interactions between minerals of the concentrate during the biooxidation. The results can be used as the basis for the development of the technologies for production of grade copper concentrate not containing zinc from sulfide copper-zinc concentrate obtained from refractory ores.