Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates

Microorganisms are used in large-scale heap or tank aeration processes for the commercial extraction of a variety of metals from their ores or concentrates. These include copper, cobalt, gold and, in the past, uranium. The metal solubilization processes are considered to be largely chemical with the microorganisms providing the chemicals and the space (exopolysaccharide layer) where the mineral dissolution reactions occur. Temperatures at which these processes are carried out can vary from ambient to 80°C and the types of organisms present depends to a large extent on the process temperature used. Irrespective of the operation temperature, biomining microbes have several characteristics in common. One shared characteristic is their ability to produce the ferric iron and sulfuric acid required to degrade the mineral and facilitate metal recovery. Other characteristics are their ability to grow autotrophically, their acid-tolerance and their inherent metal resistance or ability to acquire metal resistance. Although the microorganisms that drive the process have the above properties in common, biomining microbes usually occur in consortia in which cross-feeding may occur such that a combination of microbes including some with heterotrophic tendencies may contribute to the efficiency of the process. The remarkable adaptability of these organisms is assisted by several of the processes being continuous-flow systems that enable the continual selection of microorganisms that are more efficient at mineral degradation. Adaptability is also assisted by the processes being open and non-sterile thereby permitting new organisms to enter. This openness allows for the possibility of new genes that improve cell fitness to be selected from the horizontal gene pool. Characteristics that biomining microorganisms have in common and examples of their remarkable adaptability are described.     

Growth media in anaerobic fermentative processes: The underestimated potential of thermophilic fermentation and anaerobic digestion

Fermentation and anaerobic digestion of organic waste and wastewater is broadly studied and applied. Despite widely available results and data for these processes, comparison of the generated results in literature is difficult. Not only due to the used variety of process conditions, but also because of the many different growth media that are used. Composition of growth media can influence biogas production (rates) and lead to process instability during anaerobic digestion. To be able to compare results of the different studies reported, and to ensure nutrient limitation is not influencing observations ascribed to process dynamics and/or reaction kinetics, a standard protocol for creating a defined growth medium for anaerobic digestion and mixed culture fermentation is proposed. This paper explains the role(s) of the different macro- and micronutrients, as well as the choices for a growth medium formulation strategy. In addition, the differences in nutrient requirements between mesophilic and thermophilic systems are discussed as well as the importance of specific trace metals regarding specific conversion routes and the possible supplementary requirement of vitamins. The paper will also give some insight into the bio-availability and toxicity of trace metals. A remarkable finding is that mesophilic and thermophilic enzymes are quite comparable at their optimum temperatures. This has consequences for the trace metal requirements of thermophiles under certain conditions. Under non-limiting conditions, the trace metal requirement of thermophilic systems is about 3 times higher than for mesophilic systems.     

Mineral-microbe interactions: biotechnological potential of bioweathering

Mineral-microbe interaction has been a key factor shaping the lithosphere of our planet since the Precambrian. Detailed investigation has been mainly focused on the role of bioweathering in biomining processes, leading to the selection of highly efficient microbial inoculants for the recovery of metals. Here we expand this scenario, presenting additional applications of bacteria and fungi in mineral dissolution, a process with novel biotechnological potential that has been poorly investigated. The ability of microorganisms to trigger soil formation and to sustain plant establishment and growth are suggested as invaluable tools to counteract the expansion of arid lands and to increase crop productivity. Furthermore, interesting exploitations of mineral weathering microbes are represented by biorestoration and bioremediation technologies, innovative and competitive solutions characterized by economical and environmental advantages. Overall, in the future the study and application of the metabolic properties of microbial communities capable of weathering can represent a driving force in the expanding sector of environmental biotechnology.     

Discrimination of mesophilic and thermophilic proteins using machine learning algorithms

Discriminating thermophilic proteins from their mesophilic counterparts is a challenging task and it would help to design stable proteins. In this work, we have systematically analyzed the amino acid compositions of 3075 mesophilic and 1609 thermophilic proteins belonging to 9 and 15 families, respectively. We found that the charged residues Lys, Arg, and Glu as well as the hydrophobic residues, Val and Ile have higher occurrence in thermophiles than mesophiles. Further, we have analyzed the performance of different methods, based on Bayes rules, logistic functions, neural networks, support vector machines, decision trees and so forth for discriminating mesophilic and thermophilic proteins. We found that most of the machine learning techniques discriminate these classes of proteins with similar accuracy. The neural network-based method could discriminate the thermophiles from mesophiles at the five-fold cross-validation accuracy of 89% in a dataset of 4684 proteins. Moreover, this method is tested with 325 mesophiles in Xylella fastidosa and 382 thermophiles in Aquifex aeolicus and it could successfully discriminate them with the accuracy of 91%. These accuracy levels are better than other methods in the literature and we suggest that this method could be effectively used to discriminate mesophilic and thermophilic proteins.     

Diversity and variability of methanogens during the shift from mesophilic to thermohilic conditions while biogas production

Anaerobic digestion (AD) is the most popular path of organic waste disposal. It is often used in wastewater treatment plants for excessive sludge removal. Methanogenic fermentation had usually been performed under mesophilic conditions, but in the past few years the thermophilic processes have become more popular due to economics and sludge sanitation. Methanogens, the group of microorganisms responsible for methane production, are thought to be sensitive to temperature change and it has already been proven that the communities performing methanogenesis under mesophilic and thermophilic conditions differ. But in most cases the research performed on methanogen diversity and changeability was undertaken in two separate anaerobic chambers for meso- and thermophilic conditions. It is also known that there is a group of microorganisms performing AD which are insensitive to temperature. Also the linkage between digester performance and its microbial content and community changeability is still not fully understood. That is why in this experiment we analyzed the bacterial community performing methanogenesis in a pilot scale anaerobic chamber during the shift from mesophilic to thermophilic conditions to point at the group of temperature tolerant microorganisms and their performance. The research was performed with PCR–DGGE (polymerase chain reaction–denaturing gradient gel electrophoresis). It occurred that the community biodiversity decreased together with a temperature increase. The changes were coherent for both the total bacteria community and methanogens. These bacterial shifts were also convergent with biogas production—it decreased in the beginning of the thermophilic phase with the bacterial biodiversity decrease and increased when the community seemed to be restored. DGGE results suggest that among a wide variety of microorganisms involved in AD there is a GC-rich group relatively insensitive towards temperature change, able to adapt quickly to shifts in temperature and perform AD effectively. The studies of this microbial group could be a step forward in developing more efficient anaerobic digestion technology.     

Thermophilic organisms as sources of thermostable enzymes

Thermostable enzymes are receiving considerable attention and there are many industrial applications already. Most thermostable enzymes on the market have, however, been derived from mesophiles. Further research, especially into the possibilities of cloning enzymes from thermophiles into mesophilic hosts, will greatly increase the exploitation of thermophiles in biotechnology.     

Metal resistance in acidophilic microorganisms and its significance for biotechnologies

Extremely acidophilic microorganisms have an optimal pH of <3 and are found in all three domains of life. As metals are more soluble at acid pH, acidophiles are often challenged by very high metal concentrations. Acidophiles are metal-tolerant by both intrinsic, passive mechanisms as well as active systems. Passive mechanisms include an internal positive membrane potential that creates a chemiosmotic gradient against which metal cations must move, as well as the formation of metal sulfate complexes reducing the concentration of the free metal ion. Active systems include efflux proteins that pump metals out of the cytoplasm and conversion of the metal to a less toxic form. Acidophiles are exploited in a number of biotechnologies including biomining for sulfide mineral dissolution, biosulfidogenesis to produce sulfide that can selectively precipitate metals from process streams, treatment of acid mine drainage, and bioremediation of acidic metal-contaminated milieux. This review describes how acidophilic microorganisms tolerate extremely high metal concentrations in biotechnological processes and identifies areas of future work that hold promise for improving the efficiency of these applications.     

Thermophilic prokaryotes from deep subterranean habitats

The deep continental biosphere consists of geologically isolated ecosystems differing in their physicochemical, geological, and trophic parameters. Most of the deep ecosystems exist at elevated temperatures (50–120°C), which favor the development of thermophilic microorganisms. In many cases, the indigenous nature of subsurface microorganisms is questionable due to problems of collecting representative and non-contaminated samples. In spite of the numerous studies on the deep biosphere microbial communities, the number of cultivated thermophiles isolated from subsurface environments not associated with petroleum deposits does not exceed 30 species. More than half of the thermophilic species isolated from deep subsurface belong to the Firmicutes. The majority of the underground thermophiles are represented by strict or facultative anaerobes, with capacity for sulfate and iron reduction notably widespread. Most thermophilic subsurface microorganisms are organotrophs, although chemolithoautotrophic thermophiles also have been reported. This review deals with the phylogenetic diversity and physiological properties of the cultivated thermophilic prokaryotes isolated from various deep subterranean habitats.     

Temperature range variants of Bacillus megaterium

Facultatively and obligately thermophilic variants were isolated from 3 out of 12 tested mesophilic Bacillus megaterium strains. The variants occurred at a frequency of 10^-8–10^-9. The ability to grow at elevated temperatures was cured by means of treatment with acridine orange. Stable revertants were isolated from facultatively and obligately thermophilic variants. An unknown type of megacin was produced by the facultative thermophiles. This megacin attacked mesophilic and obligately thermophilic strains. The thermophiles displayed a few divergent taxonomic characteristics but a close relationship between the strains was indicated by the megacin spectrum and sensitivity to phage. Arrhenius plots revealed that the strains could be considered as temperature range variants and that the temperature characteristic increased with growth at a higher temperature range. The case for a plasmid involvement in the phenomenon is discussed.Abbreviations M Mesophilic - Fp facultatively psychrophilic - Ft facultatively thermophilic - Ot obligately thermophilic     

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).     

Structural differences between thermophilic and mesophilic membrane proteins

The evolutionary adaptations of thermophilic water‐soluble proteins required for maintaining stability at high temperature have been extensively investigated. Little is known about the adaptations in membrane proteins, however. Here, we compare many properties of mesophilic and thermophilic membrane protein structures, including side‐chain burial, packing, hydrogen bonding, transmembrane kinks, loop lengths, hydrophobicity, and other sequence features. Most of these properties are quite similar between mesophiles and thermophiles although we observe a slight increase in side‐chain burial and possibly a slight decrease in the frequency of transmembrane kinks in thermophilic membrane protein structures. The most striking difference is the increased hydrophobicity of thermophilic transmembrane helices, possibly reflecting more stringent hydrophobicity requirements for membrane partitioning at high temperature. In agreement with prior work examining transmembrane sequences, we find that thermophiles have an increase in small residues (Gly, Ala, Ser, and Val) and a strong suppression of Cys. We also find a relative dearth of most strongly polar residues (Asp, Asn, Glu, Gln, and Arg). These results suggest that in thermophiles, there is significant evolutionary pressure to offload destabilizing polar amino acids, to decrease the entropy cost of side chain burial, and to eliminate thermally sensitive amino acids.     

嗜热古菌异化型硫氧化途径的研究进展

微生物参与的还原性无机硫化合物的氧化过程是硫地球化学循环的重要组成部分,也可应用于生物冶金工业及酸性矿水治理等方面。嗜热古菌是高温环境中存在的一群特殊的微生物,其所参与的异化型硫氧化途径复杂多样,涉及众多氧化还原酶以及硫转运蛋白。本文将结合我们的研究工作,就参与异化型硫氧化代谢过程的嗜热古菌的种类,以及它们所参与的硫氧化过程进行系统的介绍。     

Genomics, metagenomics and proteomics in biomining microorganisms

The use of acidophilic, chemolithotrophic microorganisms capable of oxidizing iron and sulfur in industrial processes to recover metals from minerals containing copper, gold and uranium is a well established biotechnology with distinctive advantages over traditional mining. A consortium of different microorganisms participates in the oxidative reactions resulting in the extraction of dissolved metal values from ores. Considerable effort has been spent in the last years to understand the biochemistry of iron and sulfur compounds oxidation, bacteria-mineral interactions (chemotaxis, quorum sensing, adhesion, biofilm formation) and several adaptive responses allowing the microorganisms to survive in a bioleaching environment. All of these are considered key phenomena for understanding the process of biomining. The use of genomics, metagenomics and high throughput proteomics to study the global regulatory responses that the biomining community uses to adapt to their changing environment is just beginning to emerge in the last years. These powerful approaches are reviewed here since they offer the possibility of exciting new findings that will allow analyzing the community as a microbial system, determining the extent to which each of the individual participants contributes to the process, how they evolve in time to keep the conglomerate healthy and therefore efficient during the entire process of bioleaching.     

Role of cation-pi interactions to the stability of thermophilic proteins

Elucidating the factors responsible for exhibiting extreme thermal stability of thermophilic proteins is very important for an understanding of the mechanism of protein stability, as well as to design stable proteins. In this work, we have analyzed the influence of cation-pi interactions to enhance the stability from mesophilic to thermophilic proteins. The favorable residue pairs forming such a system of interactions have been brought out. We found that the Tyr has a greater number of such interactions with Lys in thermophilic proteins. Specifically, the same Lys would experience a greater number of cation-pi interactions with several Tyr residues in thermophiles. On the other hand, the influence of Phe in making cation-pi interactions is higher in mesophiles than in thermophiles. Further, a network of cation-pi interactions are maintained by Lys in thermophiles, whereas Arg plays a major role in mesophilic proteins. Moreover, atoms that have a substantial positive charge in both Lys and Arg make a more significant contribution for cation-pi interactions than do cationic group atoms.     

Microbiology of synthesis gas fermentation for biofuel production

A significant portion of biomass sources like straw and wood is poorly degradable and cannot be converted to biofuels by microorganisms. The gasification of this waste material to produce synthesis gas (or syngas) could offer a solution to this problem, as microorganisms that convert CO and H2) (the essential components of syngas) to multicarbon compounds are available. These are predominantly mesophilic microorganisms that produce short-chain fatty acids and alcohols from CO and H2. Additionally, hydrogen can be produced by carboxydotrophic hydrogenogenic bacteria that convert CO and H2O to H2 and CO2. The production of ethanol through syngas fermentation is already available as a commercial process. The use of thermophilic microorganisms for these processes could offer some advantages; however, to date, few thermophiles are known that grow well on syngas and produce organic compounds. The identification of new isolates that would broaden the product range of syngas fermentations is desirable. Metabolic engineering could be employed to broaden the variety of available products, although genetic tools for such engineering are currently unavailable. Nevertheless, syngas fermenting microorganisms possess advantageous characteristics for biofuel production and hold potential for future engineering efforts.     

Thermophilic aerobic wastewater treatment,process performance,biomass characteristics,and effluent quality

Thermophilic aerobic wastewater treatment is reviewed. Thermophilic processes have been studied in laboratory and pilot-scale while full-scale applications are rare. The paper focuses on the microbiology of aerobic thermophiles, performance of the aerobic wastewater treatments, sludge yield, and alternatives to enhance performance of thethermophilic process. Thermophilic processes have been shown to operate under markedly high loading rates (30–180 kg COD m⁻³d⁻¹).Reported sludge production values under thermophilic conditions vary between 0.05 and0.3 kg SS kg COD_removed, which are about the same or lower than generally obtained in mesophilic processes. Compared to analogous mesophilic treatment, thermophilic treatment commonly suffers from poorer effluent quality, measured by lower total COD and filtrated (GF-A) COD removals. However, in the removal of soluble (bacterial membrane filtered) COD both mesophilic and thermophilic treatments have produced similar results. Sludge settle ability in thermophilic processes have been reported to be better or poorer than in analogous mesophilic processes, although cases with better settling properties are rare. Combining thermophilic with mesophilic treatment or ultrafiltration may in some cases markedly improve effluent quality. This revised version was published online in June 2006 with corrections to the Cover Date.     

From mesophilic to thermophilic conditions: one-step temperature increase improves the methane production of a granular sludge treating agroindustrial effluents

Objectives

To assess the effect of one-step temperature increase, from 35 to 55 °C, on the methane production of a mesophilic granular sludge (MGS) treating wine vinasses and the effluent of a hydrogenogenic upflow anaerobic sludge blanket (UASB) reactor.

Results

One-step temperature increase from mesophilic to thermophilic conditions improved methane production regardless of the substrate tested. The biomethane potentials obtained under thermophilic conditions were 1.8–2.9 times higher than those obtained under mesophilic conditions. The MGS also performed better than an acclimated thermophilic digestate, producing 2.2–2.5 times more methane than the digestate under thermophilic conditions. Increasing the temperature from 35 to 55 °C also improved the methane production rate of the MGS (up to 9.4 times faster) and reduced the lag time (up to 1.9 times). Although the temperature increase mediated a decrease in the size of the sludge granules, no negative effects on the performance of the MGS was observed under thermophilic conditions.

Conclusions

More methane is obtained from real agroindustrial effluents at thermophilic conditions than under mesophilic conditions. One-step temperature increase (instead of progressive sequential increases) can be used to implement the thermophilic anaerobic digestion processes with MGS.

     

Extremozyme

Extremozymes for biotechnological applications Industrial biotechnology is a fast growing and proliferating field of research. Biocatalysis gradually replaces chemical processes and is widely used in textile or food industry or in the sustainable production of fine chemicals. Although currently most of the enzymes in industry are of mesophilic origin, the focus is changing towards more robust biocatalysts from extremophilic organisms. Research on extremophiles will progressively supply novel extremozymes for biotechnological applications. In particular (hyper‐)thermophiles, acidophiles or salt‐tolerant microorganisms are a rich source of industrial applicable and robust extremozymes with optimal activity under harsh conditions.     

Amino acid coupling patterns in thermophilic proteins

Structural analysis is useful in elucidating structural features responsible for enhanced thermal stability of proteins. However, due to the rapid increase of sequenced genomic data, there are far more protein sequences than the corresponding three-dimensional (3D) structures. The usual sequence-based amino acid composition analysis provides useful but simplified clues about the amino acid types related to thermal stability of proteins. In this work, we developed a statistical approach to identify the significant amino acid coupling sequence patterns in thermophilic proteins. The amino acid coupling sequence pattern is defined as any 2 types of amino acids separated by 1 or more amino acids. Using this approach, we construct the rho profiles for the coupling patterns. The rho value gives a measure of the relative occurrence of a coupling pattern in thermophiles compared with mesophiles. We found that thermophiles and mesophiles exhibit significant bias in their amino acid coupling patterns. We showed that such bias is mainly due to temperature adaptation instead of species or GC content variations. Though no single outstanding coupling pattern can adequately account for protein thermostability, we can use a group of amino acid coupling patterns having strong statistical significance (p values < 10(-7)) to distinguish between thermophilic and mesophilic proteins. We found a good correlation between the optimal growth temperatures of the genomes and the occurrences of the coupling patterns (the correlation coefficient is 0.89). Furthermore, we can separate the thermophilic proteins from their mesophilic orthologs using the amino acid coupling patterns. These results may be useful in the study of the enhanced stability of proteins from thermophiles-especially when structural information is scarce. Proteins 2005. (c) 2005 Wiley-Liss, Inc.