Thermophilic Culture Research Articles

Effects of Starter Culture Combination on the Characteristic of White Cheese

In this study, the effects of using mesophilic and thermophilic cheese cultures at different ratios on White cheese yield, physico-chemical, textural, sensory and microbiological properties were investigated. Yeast, mold, Enterobacter spp., and Staphylococcus aureus counts were determined. In sample with high ratio of thermophilic culture, there was slightly increase in fat, salt, fat and salt in dry matter content. All textural parameters were found significantly different. The number of Enterobacter spp. was found lower in cheese with high ratio of thermophilic culture, while the number of yeast was high. As a result, it has been observed that starter culture ratios, which contain different bacterial strains, can affect the technological and functional properties of freshly consumed cheese.

Adaptation of anaerobic culture to bioconversion of carbon dioxide with hydrogen to biomethane

Biogas is composed of energetically useable methane (CH4) ranging from 50 to 70% and carbon dioxide (CO2) taking up 30–50%. To upgrading biogas to biomethane for better exploitation of its energetical potential, many different physico-chemical processes are commercially applied. Besides these methods, biological methods for the conversion of CO2 with electrolytic H2 to biomethane based on the activity of hydrogenotrophic methanogens are also currently being researched. This study deals with the adaptation of anaerobic culture to H2 and CO2 as substrates under thermophilic and mesophilic conditions. Thermophilic cultures initially showed slower adaptation, but CO2 conversion efficiency was finally by 12.7% higher compared to the mesophilic culture under the same conditions of the H2 loading rate. Thermophilic conditions were favourable also for homoacetogens producing higher concentration of acetate from CO2 and H2 under thermophilic condition compared to mesophilic condition in the excess of H2. Maximum capacity of the used thermophilic suspended biomass for bioconversion of CO2 was the H2 loading rate 2 L/(L.d) resulting in CO2 bioconversion efficiency of 84% (median) in contrast with 40% (median) for mesophilic culture. The system was limited by the concentration of biomass round 4 g/L.

Effects of pH value on the expression of key iron/sulfur oxidation genes during bioleaching of chalcopyrite on thermophilic condition

Physicochemical factors such pH value would affect the microbial metabolism during chalcopyrite bioleaching. To this end, the effects of pH on the expression of critical functional genes during bioleaching were evaluated. A mixed culture of moderate thermophiles was used for chalcopyrite bioleaching at initial pH values of 1.0, 2.0, and 3.0, and bioleaching processes were monitored via measuring the physicochemical parameters. Quantitative real-time PCR assay was used to monitor the dynamics of microbial community structures and the expression of critical iron/sulfur oxidation genes (4Fe-4S ferredoxin and sulfate adenylyltransferase genes, respectively). Redundancy analysis and calculation of correlation coefficients were used to reveal linkages between gene expression and various physicochemical factors. The leaching processes at initial pH of 1.0 and 3.0 were prolonged compared with that at initial pH of 2.0. It was shown that Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus were the dominant species during the early stage in free and attached cells, respectively, while Ferroplasma thermophilum became predominant in the later phase. The gene expression in Sulfobacillus thermosulfidooxidans and Ferroplasma thermophilum was greatly affected by pH values. On the other hand, the relationship between pH and gene expression in Acidithiobacillus caldus was not significant. The study unraveled the importance of pH value on chalcopyrite bioleaching, and pH selectively influenced the expression of key functional genes of some specific species.

A Cryptic Non-Inducible Prophage Confers Phage-Immunity on the Streptococcus thermophilus M17PTZA496.

Streptococcus thermophilus is considered one of the most important species for the dairy industry. Due to their diffusion in dairy environments, bacteriophages can represent a threat to this widely used bacterial species. Despite the presence of a CRISPR-Cas system in the S. thermophilus genome, some lysogenic strains harbor cryptic prophages that can increase the phage-host resistance defense. This characteristic was identified in the dairy strain S. thermophilus M17PTZA496, which contains two integrated prophages 51.8 and 28.3 Kb long, respectively. In the present study, defense mechanisms, such as a lipoprotein-encoding gene and Siphovirus Gp157, the last associated to the presence of a noncoding viral DNA element, were identified in the prophage M17PTZA496 genome. The ability to overexpress genes involved in these defense mechanisms under specific stressful conditions, such as phage attack, has been demonstrated. Despite the addition of increasing amounts of Mitomycin C, M17PTZA496 was found to be non-inducible. However, the transcriptional activity of the phage terminase large subunit was detected in the presence of the antagonist phage vB_SthS-VA460 and of Mitomycin C. The discovery of an additional immune mechanism, associated with bacteriophage-insensitive strains, is of utmost importance, for technological applications and industrial processes. To our knowledge, this is the first study reporting the capability of a prophage integrated into the S. thermophilus genome expressing different phage defense mechanisms. Bacteriophages are widespread entities that constantly threaten starter cultures in the dairy industry. In cheese and yogurt manufacturing, the lysis of Streptococcus thermophilus cultures by viral attacks can lead to huge economic losses. Nowadays S. thermophilus is considered a well-stablished model organism for the study of natural adaptive immunity (CRISPR-Cas) against phage and plasmids, however, the identification of novel bacteriophage-resistance mechanisms, in this species, is strongly desirable. Here, we demonstrated that the presence of a non-inducible prophage confers phage-immunity to an S. thermophilus strain, by the presence of ltp and a viral noncoding region. S. thermophilus M17PTZA496 arises as an unconventional model to study phage resistance and potentially represents an alternative starter strain for dairy productions.

Synchrotron Radiation Based Study of the Catalytic Mechanism of Ag+ to Chalcopyrite Bioleaching by Mesophilic and Thermophilic Cultures

The catalytic mechanism of Ag+ for chalcopyrite bioleaching by mesophilic culture (at 30 °C) and thermophilic culture (at 48 °C) was investigated using synchrotron radiation-based X-ray diffraction (SR-XRD) and S K-edge and Fe L-edge X-ray absorption near edge structure (XANES) spectroscopy. Bioleaching experiments showed that copper extraction from chalcopyrite bioleaching by both cultures was promoted significantly by Ag+, with more serious corrosion occurring on the minerals surface. SR-XRD and XANES analyses showed that the intermediates S0, jarosite and secondary minerals (bornite, chalcocite and covellite) formed for all bioleaching experiments. For these secondary minerals, the formation of bornite and covellite was promoted significantly in the presence of Ag+ for both cultures, while Ag+ has almost no effect on the formation of chalcocite. These results provided insight into the catalytic mechanisms of Ag+ to chalcopyrite bioleaching by the mesophilic and thermophilic cultures, which are both probably due to the rapid formation of bornite by Ag+ and the conversion of bornite to covellite.

Influence of X-ray μ-Computed Tomography on the microbial activity of a mixed thermophilic and mesophilic bioleaching culture colonising a mineral surface

X-ray micro-Computed Tomography (X-ray μCT) allows for non-invasive 3D investigations of heap leaching processes. It permits study of mineral liberation, pore network structure in particles or beds, and bioleaching of non-surface mineral grains. A premise for bioleaching studies integrating X-ray μCT is that the metabolic activity of microorganisms is not affected by X-ray energy doses. This study therefore evaluates the compatibility of bioleaching microorganisms with X-ray energy exposure. The X-ray effect on a mixed thermophilic culture suspended in solution was assessed using imaging conditions of 35–150 kV, 200–280 μA and 1–2 hours. Microorganisms were unaffected between 35–90 kV and 200–280 μA while there was some detrimental effect at 120 and 150 kV. The X-ray influence on a mixed mesophilic culture colonising a mineral surface was investigated at 90 kV and 225 μA for a 1 h exposure time. The system was monitored before and after exposure through measurement of: redox potential of the effluent solution, microbial cell concentration, heat output from the colonised surface using isothermal microcalorimetry (IMC), and cell attachment on the ore surface using scanning electron microscopy (SEM). The redox and cell concentration measurements showed insignificant differences between exposed and unexposed samples. Equivalent heat generation and good colonization of the mineral surface was demonstrated, confirming continued microbial activity post exposure, hence recommending its use in support of bioleaching studies.

Two-step biohydrometallurgical technology of copper-zinc concentrate processing as an opportunity to reduce negative impacts on the environment

Polymetallic concentrates obtained during ore beneficiation pose a significant problem for the mining and metallurgy industry due to an increase in load on subsequent comminution steps and a high loss of metals in slag during smelting. Storage of such slag can lead to pollution of groundwater due to weathering. Biohydrometallurgy is an option for the processing of sulfidic raw materials that has a low impact on the environment. Processing of sulfidic concentrates of copper-zinc ore via bioleaching techniques was studied in this paper. Three mixed microbial cultures of acidophilic microorganisms were enriched from industrial mining sites: two autotrophic mesophilic cultures containing Acidithiobacillus ferroxidans and Leptospirillum spp. (grown at 30 and 35 °C), and a mixotrophic moderate thermophilic culture containing Sulfobacillus thermotolerans, Leptospirillum ferriphilum, as well as the archaea Ferroplasma acidiphilum and Acidiplasma spp. (grown at 40 °C). The autotrophic microbial culture growing at 30 °C was used to generate an iron-containing biosolution for ferric leaching of a copper-zinc concentrate. Zinc and iron extracted into solution faster than copper during high-temperature (80 °C) ferric leaching of the concentrate due to galvanic interactions between minerals, redox conditions of the medium, and differences between mineral oxidation mechanisms. Weight loss of the leach residue was 34.0%, with relative copper content increased by 1.0%, zinc content decreased by 6.18%, and iron content decreased by 15.1%. Biooxidation of ferrous iron in the pregnant leach solution by three microbial cultures was also studied. The most effective culture was moderate thermophilic. The results of studies on the bioregeneration of leaching solutions are relevant to the development of a two-step biohydrometallurgical technology for processing of copper-zinc concentrate with a closed cycle of technological flows. The ferrous iron biooxidation rate by the moderate thermophilic culture reached 20 g L−1 day−1. The leach residue obtained can be considered a high-grade copper concentrate able to be processed via smelting. This bioleaching process would make it possible to reduce pollution of groundwater by some toxic metals stored in slags. An environmentally friendly technology flow sheet for copper-zinc sulfidic ore processing using two-step bioleaching treatment was proposed.

Cu-state evolution during leaching of bornite at 50 °C

Abstract The bioleaching of bornite with mixed moderately thermophilic culture at 50 °C was investigated. The intermediary species formed during the leaching of bornite were characterized by XRD and XPS. In addition, the evolution of Cu-state during leaching of bornite was further studied by applying φh-pH diagram and cyclic voltammetry. The results showed that the bornite was more likely to be leached at high redox potential. Furthermore, the intermediary sulfides, such as isocubanite, covellite, chalcopyrite, disulfide, and polysulfide, were formed in the course of bornite dissolution. The Cu 2p photoelectron spectrum revealed that the valence of copper in bornite and intermediary sulfide formed in the dissolution of bornite is +1. The bornite and chalcopyrite can be converted into each other, and both can be further converted to covellite and/or chalcocite.

Two-step processing of refractory gold-containing sulfidic concentrate via biooxidation at two temperatures

A practical problem in the biohydrometallurgical processing of sulfide raw materials is the need to increase the efficiency and stability. The heating of pulp in bioreactors causes exothermic lysis of microorganisms and cessation of the production process. To find solutions to these problems, the present study examines the biooxidation of a gold-bearing sulfidic concentrate in a two-step (ferric leaching + biooxidation) process with different temperatures in the bioreactors at the biological step: 50 °C in the first bioreactor and 39 °C in the second. The main minerals of the flotation concentrate were pyrrhotite, pyrite, arsenopyrite, and stibnite. The preliminary ferric leaching of the concentrate for 5 h at 80 °C and pH 1.15–1.35 allowed oxidization of 26.8% of arsenopyrite and 54.4% of sulfur in the sulfides. At the biological step, a moderately thermophilic culture of acidophilic microorganisms dominated by representatives of the genus Sulfobacillus was used. The residence time of the pulp in each bioreactor was 2 days with a total residence time of 4 days. This study indicated that the efficiency of two-step process with the use of a two-temperature mode is comparable with the constant temperature mode and much higher than that of the one-step process. The final oxidation levels of arsenopyrite and sulfidic sulfur for the two-step process with elevated temperature in the primary bioreactor were 73.1 and 65.9%, respectively, with 82% gold recovery.

Relationships between galvanic interaction, copper extraction and community dynamics during bioleaching of chalcopyrite by a moderately thermophilic culture

A moderately thermophilic culture was enriched and adapted to bioleach chalcopyrite at high pulp density. In order to further improve copper extraction, effects of galvanic interaction on bioleaching performance and community dynamics were investigated by adding pyrite. Copper extractions were improved by 2.91 and 1.97 times in the initial and the final stages when pyrite was present, respectively. However, it did not benefit chalcopyrite dissolution in the middle stage. Community dynamics analysis showed that succession of the attached cells was significantly different from community dynamics of the planktonic cells. One of planktonic populations always dominated the communities in most cases, while no species had absolutely competitive advantages in the attached communities. In addition, the presence of pyrite had significant effects on planktonic and attached community structures, and could accelerate planktonic community succession.

Bioregeneration of Leaching Solutions during Two-Step Processing of Copper-Zinc Concentrate

A comparative study of the oxidation of ferrous iron ions by various cultures of acidophilic chemolithotrophic microorganisms in solutions obtained after ferric leaching of copper-zinc concentrate at 80°C has been carried out. It was shown that the use of a moderately thermophilic culture for bioregeneration of leaching solutions was preferable. At the same time, the oxidation rate of Fe2+ ions reached 0.88 g/(L h), or 21.1 g/(L day). We propose that the activity of the moderately thermophilic culture was due to the presence of the mixotrophic bacteria Sulfobacillus spp., which used organic products of the microbial lysis for their growth. These products were formed during high-temperature ferric leaching of the copper-zinc concentrate with the biosolution.

Co-Bioleaching of Chalcopyrite and Silver-Bearing Bornite in a Mixed Moderately Thermophilic Culture

Chalcopyrite and bornite are two important copper minerals, and they often coexist. In this study, the co-bioleaching of chalcopyrite and silver-bearing bornite by mixed moderately thermophilic culture at 50 °C was investigated. The bioleaching results show that the extraction percentage of Cu for co-bioleaching of chalcopyrite (Ccp) and silver-bearing bornite (Bn) (Ccp/Bn = 3:1) was 94.6%. Compared to bioleaching of chalcopyrite or silver-bearing bornite alone, the Cu extraction percentage was greatly enhanced when they were bioleached together. The leaching residues were characterized by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). Silver-bearing bornite dissolved preferentially compared to chalcopyrite, due to galvanic interactions. Simultaneously, Ag+ was released from the silver-bearing bornite into solution. Ag2S formed on the surface because Cu and Fe in the chalcopyrite were replaced by Ag+, accelerating chalcopyrite dissolution and enrichment of Ag on the surface of the chalcopyrite.

Preservation of thermophilic mixed culture for anaerobic palm oil mill effluent treatment by convective drying methods

The generation of huge amount of liquid waste known as palm oil mill effluent is a major problem in oil palm industry. Meanwhile, anaerobic biodegradation of such organic effluent at thermophilic condition is a promising treatment technology due to its high efficiency. However, storage and transportation of thermophilic mixed culture sludge are challenging due to constant biogas generation and heating requirement. Hence, drying of thermophilic sludge was conducted to obtain dormant thermophiles and thus enables easier handling. In this study, thermophilic sludge was dried using heat pump at 22 and 32 °C as well as hot air oven at 40, 50, 60, and 70 °C. Subsequently, quality of dried sludge was examined based on most probable number enumeration, chemical oxygen demand, and methane yield. Average drying rate was found to increase from 3.21 to 17.84 g H2O/m2 min as drying temperatures increases while average moisture diffusivity values ranges from 5.07 × 10−9 to 4.34 × 10−8 m2/s. Oven drying of thermophilic mixed culture resulted in highest chemical oxygen demand removal and lowest log reduction of anaerobes at 53.41% and 2.16, respectively, while heat pump drying resulted in the highest methane yield and lowest log reduction of methanogens at 53.4 ml CH4/g COD and 2.09, respectively. To conclude, heat pump at 22 °C was most suitable drying technique for thermophilic mixed culture as the original methane-producing capability was largely retained after drying, at a slightly lower yet still comparable chemical oxygen demand removal when palm oil mill effluent was treated with the rehydrated culture.

Biooxidation of a High-Grade Arsenopyritic Gold Ore Using a Mixed Culture of Moderate Thermophilic Microorganisms

This study investigated the biooxidation of a high-grade refractory gold ore from the Zarshouran mine (West Azarbaijan, Iran) in shake flasks and a stirred tank bioreactor (STBR) using a mixed culture of moderately thermophilic microorganisms. The influence of four critical parameters including, pH, biooxidation time, nutrient medium type and pulp density on the iron and arsenic extraction as well as gold cyanidation were evaluated in a full factorial design in shake flasks at 45 °C. Maximum iron extraction was obtained in M9K medium, pulp density of 5% (w/v), 15 days of biooxidation time and the pH of 1.6. Biooxidation in the STBR was carried out at the pulp densities of 10% and 20% (w/v), the pH of 1.7 in the M9K medium, in which dissolved oxygen, pH, redox potential, iron and arsenic concentrations were measured during the process. It was found that 61% decrement of sulphur content in the STBR led to recover 80% of gold, which was 43% higher than that in the conventional cyanidation process. It can be concluded that the oxidation of refractory arsenopyrite gold ore can achieved using a moderately thermophilic biooxidation culture.

Diversity of Thermophilic Iron-Pyrite-Oxidizing Enrichments from Solfataric Hot Springs in the Chilean Altiplano

Relatively little is known about the microbial communities present in natural environments that meet physico-chemical conditions for the development of potential leaching microorganisms such as thermal ecosystems from the Chilean Altiplano. Thermophilic leaching enrichments were obtained and identified from a high altitude solfataric pound in Lirima hot springs in the Chilean Altiplano. This ecosystem is characterized by hot underground freshwaters, enriched in sulfur compounds showing pH from neutral to acidic. Microbial diversity has been scarcely explored here, and preliminary results demonstrate that hydrothermal pounds are represented by thermophilic anaerobic and acidophilic taxa. Thermophilic leaching cultures in shake flasks were obtained using ferrous iron and pyrite as energy source. The presence of Bacteria and Archaea in oxidizing enrichments was determined by PCR amplification of 16S rRNA genes. A preliminary analysis of microbial diversity using massive sequencing revealed that Bacteria were more abundant than Archaea in both enrichments. Specifically for the iron-oxidizing culture, the majority of the sequences clustered within the Proteobacteria phylum (79%). Among Proteobacteria, the proportion of Betaproteobacteria (42.2%) and Gammaproteobacteria (21.27%) was much higher than that of Alphaproteobacteria (15.5%). Within the Betaproteobacteria class, the most frequent genus was Leptothrix-like. Similar results were obtained for the pyrite oxidizing culture. Interestingly, this study shows the presence of microorganisms close to the Leptothrix genus under low pH conditions (1.7-2.8) and their capacity to grow at high temperatures with ferrous iron or pyrite as sole energy source

Effect of X-Ray µCT Scanning on the Growth and Activity of Microorganisms in a Heap Bioleaching System

In heap bioleaching, a process in which microorganisms are required for the regeneration of leach reagents and control of reaction products, inaccessibility of non-surface mineral grains is a key cause of low recovery and long extraction times. High resolution, non-destructive 3D X-ray micro-computed tomography (μCT) is an imaging technique that has been successfully demonstrated for the study of abiotic leaching of non-surface minerals. For this technique to be applied to biotic leaching, it is required that the iron and sulphur oxidizing abilities of the microorganisms are not affected by the irradiation experienced. In the current study, the feasibility of investigating biotic leaching by X-ray μCT is explored by examining the relative energies required to achieve the high image resolution needed for mineral grain mapping while avoiding microbial deactivation. A mixed mesophilic and moderately thermophilic culture in solution was used and exposed to various X-ray energy doses. Direct microscopic cell counting and redox potential were measured to quantify the microbial activity and growth. The results showed that exposure to X-ray does not affect microbial activity at 35-90 kV, 200-280 μA and a distance of 7.2 cm between energy source and sample, however, it has an influence at 120 and 150 kV. This indicates that while X-ray μCT does influence the microbial cultures, it can be used for bioleaching studies at lower energy doses.

Recycling of metals from pretreated waste printed circuit boards effectively in stirred tank reactor by a moderately thermophilic culture

To seek a feasible technique for processing waste printed circuit boards (PCBs), pretreatment of PCBs by table separation and further bioleached by moderate thermophiles in a stirred tank reactor were investigated. The shaking table separation, conducted after grinding and sieving of PCBs, produced two fractions: metal-rich parts (RPCBs), which is more suitable for pyrometallurgy process than untreated PCBs, and metal-poor parts (PPCBs) with only 8.83% metals was then bioleached by a mixed culture of moderate thermophiles effectively. After adaptation, the mixed culture could tolerate 80g/L PPCBs. The bioleaching results showed that metals recovery was 85.23% Zn, 76.59% Cu and 70.16% Al in only 7 days. Trace Pb and Sn were detected in the leachate because of precipitating. The microorganism community structure was analyzed by amplified ribosomal DNA restriction analysis. Two moderately thermophilic bacteria species were identified as Leptospirillum ferriphilum and Acidithiobacillus caldus. Furthermore, uncultured Thermoplasmatales archaeon was also detected in the leaching system. It was also shown that moderate thermophiles revealed best bioleaching ability when compared with mesophiles and the mixture of mesophiles and moderate thermophiles. Finally, we designed a two-stage process model according to the present study to achieve semi-industrial waste PCBs recycling and economic feasibility analysis indicated that the process was profitable.

Comparative study of S, Fe and Cu speciation transformation during chalcopyrite bioleaching by mixed mesophiles and mixed thermophiles

The bioleaching experiments of chalcopyrite were conducted with mixed mesophilic culture (30°C), moderately thermophilic culture (45°C) and extremely thermophilic culture (65°C), respectively. During bioleaching, the S/Fe/Cu speciation was analyzed by synchrotron radiation (SR) based X-ray diffraction (XRD) and S, Fe and Cu K-edge X-ray absorption near edge structure (XANES) spectroscopy. The results showed that the chalcopyrite dissolution could be significantly promoted by these mixed cultures, and the promotion effects were enhanced with the increase of bioleaching temperature. For all bioleaching tests, the formation of intermediates S0, jarosite and secondary minerals (chalcocite, bornite and covellite) was detected, in which the formation of bornite, chalcocite and covellite was just related with redox potential. The formation of bornite could accelerate chalcopyrite dissolution, while the decrease of bioleaching microorganisms could reduce chalcopyrite dissolution.

A comprehensive utilization of silver-bearing solid wastes in chalcopyrite bioleaching

In this work, two types of abandoned silver-bearing solid wastes of zinc leaching residue (ZLR) and firebrick were used as silver catalysis in chalcopyrite bioleaching by mixed moderately thermophilic culture. Bioleaching results showed that the chosen ZLR and waste firebrick both remarkably promoted chalcopyrite bioleaching, and chalcopyrite was not passivated at high redox potential (more than 600mV vs. Ag/AgCl) in the presence of silver-bearing solid wastes. The bioleaching residues were subsequently leached by thiourea to recover silver. Results indicated that silver was more easily to be extracted from bioleaching residues than from untreated silver-bearing solid wastes, and high silver recovery of about 90% can be finally achieved. Hence, this work is potentially useful to provide a promising technique for promoting chalcopyrite bioleaching and for comprehensive utilization of silver-bearing solid wastes.

Selection of thermophiles for base metal sulfide concentrate leaching, Part II: Nickel-copper and nickel concentrates

The ferrous iron- and sulfur-oxidizing Acidithiomicrobium was the dominant iron-oxidizing bacterium in a mixed culture of moderate thermophiles in a bioreactor with a high concentration of nickel in solution (20gl−1) and a continuous feed of nickel concentrate. This is the first demonstration specifically indicating that this bacterium could be used for industrial processing of a base metal sulfide concentrate. However, its sensitivity to copper determined that it was replaced by Sulfobacillus thermosulfidooxidans in the mixed culture when the feed was a nickel-copper concentrate. The extraction of copper from the nickel-copper concentrate by moderate thermophiles, despite fine grinding of the feed, was relatively poor (50% at 49°C) compared to that achieved with high temperature archaea (92% at 77°C) in single, continuous reactors with 5% w/v concentrate feeds and residence times of 2.7 and 2.5days respectively.