Thermophilic Culture Research Articles

Formation of volatile compounds in Teleme cheese manufactured with mesophilic and thermophilic dairy starters

Teleme cheese from sheep, goat and cow milk was produced and the effect of three different starter cultures (mesophilic, thermophilic and mixed) on the formation of volatile compounds during ripening period up to 6 months was studied. Analysis of volatile compounds was performed by an automatic balance pressure headspace apparatus coupled to a GC/MS. The volatile fraction of Teleme cheeses displayed 27 volatile compounds including aldehydes, alcohols, ketones fatty acids and esters. The amount of total volatile compounds (except aldehydes) increased during ripening. The most abundant volatile compounds were butan-2-one, ethanol, butan-2-ol and acetic acid. The results indicated that the type of culture was found to affect to the formation of volatile compounds of the produced cheese. Cheeses made with thermophilic culture showed the highest mean levels of propanal, butanal, none-2-al, hexanal, heptanal, 3-methylbutanal, ethanol, butan-2-one, acetoin, acetone, diacetyl and acetic acid, while cheeses made with mesophilic and mixed cultures showed the lowest and intermediate levels, respectively. The type of milk used and the ripening time were also found to affect to the formation of volatile compounds. Sheep's milk cheese showed higher mean levels of acetaldehyde, propanal, butan-2-ol, 3-hydroxybutan-2-one and acetic acid, whereas the mean level of hexanal, acetone and 3-methylbutan-2-one were higher in goat cheese.

Production of novel fermented milks

The objective of this research was to test the influence of various natural substances on acid production, growth, and viability of characteristic microorganisms in yogurt and probiotic fermented dairy foods. Oligofructose, inulin, honey, and the dried biomass of Spirulina (Arthrospira) platensis were found to stimulate the growth rate and acid production activity of the major thermophilic diary cultures tested and, in addition to this, the presence of the aforementioned substrates also improved the survival of starter bacteria in fermented milk products during storage. The reduced production time of cultured milks resulted in increased production efficiency. The stimulatory and/or protective effect of oligofructose,inulin, honey, and Spirulina on Bifidobacterium spp. is probably the most important finding of this study because bifidobacteria do not grow well in milk and they have low survival rates in conventional fermented milks. Some of the bioactive substances tested were also capable of exerting an antifungal effect on spoilage yeasts and molds, and improving the nutritional and sensory properties of the final product, thus providing a new opportunity for manufacture of functional fermented dairy foods.

Effect of ammonium and acetate on methanogenic pathway and methanogenic community composition

Methanogenesis from acetate (aceticlastic methanogenesis or syntrophic acetate oxidation (SAO) coupled with hydrogenotrophic methanogenesis) is the most important step for the biogas process. The major environmental factors influencing methanogenesis are volatile fatty acids, ammonia, pH, and temperature. In our study, the effect of acetate and ammonia concentration on the methanogenic pathway from acetate and on the methanogenic communities was elucidated in two experiments: one where inocula were gradually exposed to increasing concentrations of acetate or ammonia, and another with direct exposure to different ammonia concentrations. The methanogenic pathway was determined by following the production of (14) CH(4) and (14) CO(2) from acetate labeled in the methyl group (C-2). Microbial communities' composition was determined by fluorescence in situ hybridization. Upon acclimatization to acetate and ammonia, thermophilic cultures clearly shifted their acetate bioconversion pathway from SAO with subsequent hydrogenotrophic methanogenesis (mediated by Methanobacteriales spp. and/or Methanomicrobiales spp.) to aceticlastic methanogenesis (mediated by Methanosarcinaceae spp.). On the contrary, acclimatization process resulted in no pathway shift with the mesophilic acclimatized culture. When nonacclimatized thermophilic culture was exposed to high ammonia levels (7 g NH4 +-N L(-1)), aceticlastic Methanosarcinaceae spp. was found to be the dominant methanogen.

Identification of Streptococcus thermophilus used in starter cultures in the production of hard cheeses

A prototype developed by the method of revealing Streptococcus thermophilus in PCR with the aid of synthetic oligonucleotide primers in starter cultures in the production of hard cheeses has specificity and makes it possible to effectively reveal and identify S. thermophilus strains and cultures used in the dairy processing industry.

Comparison of mesophilic and thermophilic anaerobic hydrogen production by hot spring enrichment culture

Abstract Anaerobic hydrogen producing mesophilic and thermophilic cultures were enriched and studied from an intermediate temperature (45 °C) hot spring sample. H2 production yields at 37 °C and 55 °C were highest at the initial pH of 6.5 and 7.5, respectively. Optimum glucose, iron and nickel concentrations were 9 g/l, 25 mg/l and 25 mg/l both at 37 °C and 55 °C, respectively. The highest H2 yields at 37 °C and 55 °C were 1.8 and 1.0 mol H2/mol glucose, respectively, with the optimal pH, glucose concentration and iron addition. Hydrogen production from glucose at 55 °C and 37 °C was associated with ethanol- and acetate–butyrate type fermentations, respectively. Bacterial composition was analyzed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Clostridium species dominated at both temperatures and the microbial diversity decreased with increasing temperature. At 55 °C, Clostridium ramosum was the dominant organism.

Genotyping of Streptococcus thermophilus strains isolated from traditional Egyptian dairy products by sequence analysis of the phosphoserine phosphatase (serB) gene with phenotypic characterizations of the strains

To develop a new, simplified genotyping method for examining the genetic diversity of Streptococcus thermophilus strains isolated from traditional Egyptian fermented dairy products and to characterize phenotypic traits of those strains related to their potential use in bioprocessing applications. A novel, simplified approach was developed for genotyping Strep. thermophilus involving the analysis of nucleotide sequence variations within a housekeeping gene encoding the phosphoserine phosphatase (SerB). Using this method, it was possible to identify ten genotypes involving diverse serB alleles among 54 Strep. thermophilus isolates cultured from Egyptian dairy products. These isolates harboured five de novo serB alleles that have not been detected in other Strep. thermophilus strains, deposited in a multilocus sequence typing (MLST) database. To assess distinct genotypes of the organism with phenotypic traits relevant to their potential use in industry, Strep. thermophilus strains were all subjected to a series of phenotypic characterizations. The strains were found to exhibit phenotypic diversity in terms of their ability to ferment lactose and galactose, express urease activity, produce exopolysaccharides and develop acidity. The analysis of nucleotide sequence variations within the serB gene could serve as a suitable tool for probing diverse genotypes of Strep. thermophilus. Streptococcus thermophilus isolates associated with traditional Egyptian dairy products show high degree of genetic and phenotypic diversity. This study presents a novel, simplified procedure based on serB nucleotide sequencing for genotyping Strep. thermophilus. It also provides a pool of phenotypically diverse Strep. thermophilus cultures, from which certain strains could be selected for use in bioprocessing applications including the preparation of fermented dairy products.

A conceptual flowsheet for heap leaching of platinum group metals (PGMs) from a low-grade ore concentrate

This research proposes a new conceptual process to economically extract platinum group metals (PGMs), and as a secondary aim, base metals (BMs) from a low-grade concentrate originating from typical PGM concentrator plants. Slurry made from the concentrate was coated onto granite pebbles and packed into a column, in which it was bioleached with a mixed culture of thermophiles and mesophiles at 65 °C. After 30 days the extractions achieved were 52% copper, 95% nickel and 85% cobalt. The residual concentrate material was subsequently subjected to a cyanide leach also in a packed column operating at a room temperature of 23 °C. After 21 days 20.3% Pt, 87% Pd and 46% Rh were extracted. Using these results and projected extractions over longer operating times, a conceptual flowsheet was proposed for a possible process route to recover PGM values circumventing the problematic smelter route for this material.

Effect of electromagnetic radiation of the extremely high frequency millimeter range on thermophilic cultures of bacteria of lactic acid products

The vital functions of lactic acid bacteria are investigated in three kinds of lactic acid products: with the use of Bulgaria bacillus, thermophilic streptococcus, and yogurt starter. Electromagnetic radiation in the EHF mm range at frequency 129 GHz of the mammary gland and pasteurized milk reduced the acidity of all types of products and increased the number of lactic acid bacteria in them from the first day of storage.

A comparison of bioleaching ability of mesophilic and moderately thermophilic culture on copper bioleaching from flotation concentrate and smelter dust

This study evaluates bioleaching treatments of chalcopyrite concentrate and smelter dust using mesophilic and moderately thermophilic bacterial cultures. Effects of some variable parameters such as solids concentration, temperature (type of bacteria) and inoculation on the bioleaching of the materials obtained from Sarcheshmeh copper complex were investigated. Bioleaching experiments were carried out in shaking incubator using mixed mesophilic and moderately thermoacidophilic bacteria. The results indicate that the efficiency of copper extraction is dependent on all the variables studied for both microorganisms. Maximum copper recovery from concentrate and dust was achieved using a moderately thermophilic culture. Under optimum conditions, copper dissolution from concentrate reached 87.52% with moderately thermophilic, and 34.55% with mesophilic culture, after 25 days. Also, overall copper extraction calculated for dust bioleaching were 92.00% with moderately thermophilic bacteria and 73.27% with mesophilic bacteria.

Biodegradation of polyether‐polyol‐based polyurethane elastomeric films: influence of partial replacement of polyether polyol by biopolymers of renewable origin

In this work we investigated the degradation process of polyether‐polyol‐based polyurethane (PUR) elastomeric films in the presence of a mixed thermophilic culture as a model of a natural bacterial consortium. The presence of PUR material in cultivation medium resulted in delayed but intensive growth of the bacterial culture. The unusually long lag phase was caused by the release of unreacted polyether polyol and tin catalyst from the material. The lag phase was significantly shortened and the biodegradability of PUR materials was enhanced by partial replacement (10%) of polyether polyol with biopolymers (carboxymethyl cellulose, hydroxyethyl cellulose, acetyl cellulose and actylated starch). The process of material degradation consisted of two steps. First, the materials were mechanically disrupted and, second, the bacterial culture was able to utilize abiotic degradation products, which resulted in supported bacterial growth. Direct utilization of PUR by the bacterial culture was observed as well, but the bacterial culture contributed only slightly to the total mass losses. The only exception was PUR material modified by acetyl cellulose. In this case, direct biodegradation represented the major mechanism of material decomposition. Moreover, PUR material modified by acetyl cellulose did not tend to undergo abiotic degradation. In conclusion, the modification of PUR by proper biopolymers is a promising strategy for reducing potential negative effects of waste PUR materials on the environment and enhancing their biodegradability.

Enhanced biohydrogen production from tofu residue by acid/base pretreatment and sewage sludge addition

Anaerobic dark fermentation is considered a promising technology for clean energy production and waste reduction. In the present work, tofu residue and sewage sludge were utilized as substrates for fermentative hydrogen production. To increase the biodegradability, tofu residue was pretreated for 30 min in the presence of HCl and NaOH at various concentrations (0, 0.5, 1.0, and 2.0%), and then fermented by a thermophilic (60 °C) mixed culture. The solubility (SCOD/TCOD ratio) of the tofu residue increased from 4 to 30–40% after pretreatment, and the increased soluble constituents were mainly protein rather than carbohydrate compounds. However, in spite of a slight increase of carbohydrate solubility, the H 2 production performance was significantly enhanced by pretreatment, owing to the degradability of thermophilic cultures used on insoluble tofu residues. The limited H 2 yield of 0.30 mol H 2/mol hexose added achieved in the raw tofu residue was increased 1.6- to 4-fold with the highest H 2 yield of 1.25 mol H 2/mol hexose added at 1.0% HCl concentration. Carbohydrate degradation and the H 2 production rate also increased from 39 to 50–65% and 27 to 50–120 mL H 2/L/h, respectively. The role of pretreatment was not only to increase the biodegradability but also to suppress the activity of indigenous non H 2-producers such as lactic acid bacteria and propionic acid bacteria. When sewage sludge was added to acid pretreated (1.0% HCl) tofu residue as a co-substrate, the H 2 yield and H 2 production rate increased to 1.48 mol H 2/mol hexose added and 161 mL H 2/L/h, respectively, which was attributed to the abundant minerals, vitamins, and metals contained in sewage sludge.

Tank bioleaching of low-grade chalcopyrite concentrates using redox control

Bioleaching tests were carried out in bench-scale piloting facilities, comprising fully controlled multi-stage continuously operated reactor systems using moderate thermophile and thermophile consortia under both uncontrolled and controlled redox potential conditions to assess the effect of temperature and redox control on chalcopyrite leach kinetics. The bench-scale studies demonstrated that copper recoveries > 95% could be achieved without redox control, using a thermophilic culture at 70 °C. Acceptable copper extractions could not be achieved under similar conditions at 45 °C. A significant increase in the copper leach kinetics and copper extractions was, however, achieved by controlling the redox potential in the 45 °C system at 420 mV. Final copper extractions increased from 64% at high redox potential to 97% under controlled redox conditions and as a result of the improvement in the Cu leach performance there is potential to reduce the overall residence time of the process. A key advantage of the process is that the bioleaching process costs are minimized, since the pyrite was not oxidized.

Effect of biooxidation conditions on cyanide consumption and gold recovery from a refractory gold concentrate

This study investigates the biooxidation of a refractory gold concentrate using a mixed culture of acidophilic mesophiles, moderate thermophiles and extreme thermophiles and their effect on the subsequent cyanidation and gold recovery. The experiments with high % solids using mixed mesophiles showed better oxidation potential compared to moderate thermophiles and extreme thermophiles. However, the extreme thermophiles performed better than mesophiles and moderate thermophiles during the biooxidation with < 5% solids (w/v). The biooxidized residues obtained from the experiments with extremophiles, gave a maximum gold recovery of 92% after cyanidation. The NaCN consumption during cyanidation of the biooxidized residues obtained from experiments with extremophiles was lower than bioresidues obtained from experiments with mesophiles and moderate thermophiles. The highest consumption of NaCN of 17.5 kg/ton was from the experiment with 10% solids (w/v) using mesophiles. X-ray diffraction studies and chemical analysis of the biooxidized residues confirms the precipitation of jarosites at high redox potential which produces some ferricyanide, thereby explaining the reason for high cyanide consumption during cyanidation.

A Visual Insight into the Oxidation of Sulfide Minerals During Bioleaching and Chemical Leaching of a Complex Ore

A scanning electron microscope (SEM) study was performed to provide a visual insight into the oxidation patterns of sulfide minerals during chemical and bacterial leaching of a complex ore for 3 days. The mineral grains were studied under SEM before and after bacterial and chemical leaching with or without the addition of ferrous iron to generate ferric iron in situ by bacteria or chemical oxidant (MnO2). Both mesophilic and moderately thermophilic cultures of bacteria were used in bioleaching tests. A limited oxidation of sphalerite and pyrite, similar to those in acid leaching (control), was observed to occur when no ferrous iron was added. However, the initial addition of ferrous iron into bioleaching media was shown to significantly improve the oxidation of sphalerite and pyrite. Galena was readily oxidized in the presence or absence of bacteria. Sphalerite was oxidized more extensively/selectively than chalcopyrite and pyrite, consistent with their respective nobility/electrochemical activity. Provided that chemical/biological oxidation of sphalerite was intensive, a sulfur-rich layer appeared to form on mineral surface. But, no such layer on pyrite surfaces was discernable. Supplementary bioleaching data were also provided to support SEM observations and to further elucidate the bioleaching characteristics of these sulfide phases. It can be inferred from this study that the oxidation of sulfides proceeds most discernibly via “indirect mechanism” and the generation of ferric iron by bacteria in sufficient quantity is essential for the effective oxidation of sulfide minerals.

Preservation of moderately thermophilic culture by freeze drying and frozen preservation way and effect on subsequent bioleaching of chalcopyrite

Freeze drying and frozen preservation way was used to preserve a moderately thermophilic culture for bioleaching of chalcopyrite concentrate. After preservation of 15 months, the cell viability rate decreases to 22% with a cell density of 7×10 7 mL −1. When the growth time was extended from 8 days to 14 days, cell density would increase in a large scale to about 3×10 8 mL −1. In the bioleaching experiments, unpreserved and preserved cultures were compared for dissolving chalcopyrite concentrate. Before 44 days, the unpreserved culture can reach a high copper extraction of about 17.4 g/L. While the preserved culture shows a rather low copper extraction, which is only 9.7 g/L. When the bioleaching time was extended to 80 days, copper extraction by preserved culture increases remarkably, and the concentration of copper finally achieves up to 18.3 g/L. On the other hand, copper extraction by the unpreserved culture does not show remarkable increase from 44th to the 80th day, and finally the total copper extraction is 19.8 g/L. As a result, total copper extraction in 80 days by preserved culture approaches that by unpreserved culture and freeze drying and frozen preservation even after 15 months does not bring much decrease of bioleaching ability.

Determination of microbial contamination sources for use in quality management of cheese industry: “Dil” cheese as an example

The microbiological quality, safety and shelf-life of cheeses depend on manufacture and handling in an environment that meets basic standards for hygiene and the management of hygiene in the process. In this research contamination sources of “Dil” cheese during production in a local dairy plant in Bursa, Turkey were determined. Eighteen different control points (raw milk, pasteurized milk, heated curd, molded cheese before kneading, kneaded cheese, brine solution for kneading, thermophilic culture, rennet, calcium chloride solution, brine solution for cheese, cheese vat, workers hands, production room air, production room floor, production room wall, packaging material and packaged cheese) have been examined for the enumeration of total aerobic mesophilic bacteria, Staphylococci, Enterobacteriaceae, Salmonella spp., Escherichia coli, lactic acid bacteria, Pseudomonas spp. and yeast-moulds. It was determined that viability of lactic acid bacteria in thermophilic culture was not in high numbers and some contaminations to “Dil” cheese were detected from the starter culture. Brine solutions and rennet were contaminated with Staphylococci. Yeast and moulds in production room air were the major sources of contamination. Pasteurization and kneading in hot brine solution can eliminate some of the microorganisms but that was not sufficient in the production of Dil cheese. Finish cheese should meet specific hygienic standards, with respect to regulations post-contaminations to the cheese must be inhibited and a HACCP plan should be established during production.

Thermophilic Anaerobic Digestion Using an Induced Bed Reactor

An induced bed reactor (IBR) was developed at Utah State University (USU) to apply high-rate anaerobic digestion techniques to high-suspended-solids content substrates (6% to 12% total solids) such as food waste and dairy manures. This technology has been successfully implemented at multiple full-scale installations in the U.S. and Canada as a waste treatment and energy production technology. A 58 L bench-scale reactor was operated at 55C with three organic loading rates and three corresponding hydraulic retention times using a dextrose/yeast extract substrate at 30.6 g L-1 COD. Influent and effluent streams were monitored for solids composition, gas quality and quantity, and chemical oxygen demand (COD). Residence time distribution (RTD) was determined using step input tracer studies with rhodamine WT and lithium tracers. The IBR was successfully operated at thermophilic temperatures for 180 days, demonstrating a COD removal rate of 64% and a robust thermophilic culture. Under the conditions studied, the IBR exhibits complete mix behavior with no mechanical energy input. Development of a granulated sludge bed comprising a settled sludge volume of 120 mL L-1 was evident. Further work should be done to determine conditions required for improved solids retention and thus improved performance.

Effects of high pressure treatment on glycolytic enzymes of Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Lactobacillus acidophilus

High pressure processing (HPP) reduces the glycolytic activity of lactic acid bacteria (LAB) and provides a means to control further production of acidic metabolites in fermented dairy products during storage. However, there is limited information on the effects of HPP on specific enzymes of dairy starter bacteria responsible for the metabolism of lactose. The aim of this study was to determine pressure-induced inactivation of glycolytic enzymes in Lactococcus lactis subsp. lactis C10, Streptococcus thermophilus TS1 and Lactobacillus acidophilus 2400. Cultures were grown for 16 h in M17 or MRS broth containing 5% (w/v) lactose at pH 6.5 (maintained by addition of 10 M NaOH). The cells were harvested by centrifugation, washed and resuspended in 100 mM phosphate buffer (pH 6.5) and pressure-treated at 300 and 600 MPa (≤ 22 °C, 5 min). The ability of pressure-treated resting cells of Lactococcus, incubated with 5% (w/v) lactose at 30 °C, to ferment lactose was evaluated by determining titratable acidity (TA) during incubation. The activities of phospho-β-galactosidase (P-β-gal), β-galactosidase (β-gal) and lactate dehydrogenase (LDH) were determined in cell-free extracts of untreated and pressure-treated cells. Resting cells of Lactococcus treated at 600 MPa had a substantially lower rate of acidification than the controls and those treated at 300 MPa. Both P-β-gal and β-gal were significantly inactivated (p < 0.01) in the starter cultures treated at 300 or 600 MPa. The LDH in Lactococcus and Lactobacillus was highly resistant to pressure treatment at 300 MPa. In contrast, the LDH in Streptococcus was almost completely inactivated at ≥ 300 MPa. Industrial relevance Continuing production of acidic metabolites in fermented dairy products during storage can be a technological challenge that adversely affects product quality. The current study demonstrates that high pressure processing (HPP) offers the potential of controlling this problem by inactivation of glycolytic enzymes in various mesophilic and thermophilic starter cultures. The findings of this research will assist in establishing optimised operating parameters for HPP treatment of cultured products to extend shelf-life, by reducing acid production during storage.

Biooxidation of Refractory Gold Ores by Mixed Moderate Thermophiles in Airlift Bioreactor

Bacterial preoxidation of refractory gold ore has showed many advantages, such as lower operation cost and environmental friendly. In this paper, biooxidation of refractory gold ore by mixed moderate thermophiles culture in airlift bioreactor has been well studied. Firstly, a laboratory scale airlift bioreactor unit has been designed for bacterial preoxidation of refractory gold ore, a mixed moderate thermophilic culture was incubated. The orthogonal experiment was performed to investigate the effects of particle size, pulp density, aeration rate and initial pH. The results showed that when particle size 37micrometer，pulp density 5%，aeration rate 4 dm3•s-1 and initial pH 1.2, the arsenic leaching rate achieved to 93.5%. On the base of the airlift bioreactor unit, a continuous airlift bioreactor was designed. The preoxidation of refractory gold ore by a mixed culture of moderately thermophilic bacteria also was investigated in this equipment. The results showed that, under the conditions of pulp density 8% and stay time 60h, it can deal with 0.256 kg refractory gold ore per day, and the arsenic leaching rate can reach to 95.25%. During the experiment, some leaching liquor has been sampled from the second sect the fourth column of the continuous airlift bioreactor. The composition of microbial communities in this sample has been investigated using culture-independent 16S rDNA based cloning approach- restriction fragment length polymorphism. The phylogenetic analysis revealed that Leptospirillum ferriphilum and Acidithiobacillus caldus are the main organisms in this system. Both of them are moderate thermphilic bacteria and can resist relatively high concentration of arsenic ion.

Acidianus Brierleyi is the Dominant Thermoacidophile in a Bioleaching Community Processing Chalcopyrite Containing Concentrates at 70°C

Bioleaching test work was performed in continuously operated multi-stage reactor systems at 70°C using a thermophilic culture treating an Aguablanca Ni-Cu concentrate from Spain and a blend of Cu concentrates from Bor, Serbia. The copper in both these concentrates occurs as chalcopyrite and therefore the use of thermophiles was applied, which resulted in copper recoveries of over 95%. Qualitative assessment of the microbial community in the bioreactors was performed by terminal restriction enzyme fragment length polymorphism (T-RFLP) and clone library analysis of the 16S rRNA genes amplified by PCR. T-RFLP analysis revealed that only archaea were present, and that the communities in both the Aguablanca and Bor systems consisted of two different microorganisms. A 16S rRNA gene clone library using DNA from the Aguablanca system was constructed and screened. Again, two archaea were detected in similar relative abundance in the population as found by T-RFLP analyses. The sequences of these two cloned genes revealed that the dominant archaeon (up to 98% of the total archaea detected) was Acidianus brierleyi, and the other was Metallosphaera sedula. Quantitative assessment of the microbial community was performed by Q-PCR and confirmed the dominance of archaea in the system with Acidianus being the dominant strain (98-99% of the total population) and a minor part of the population (1-2%) consisted of Metallosphaera. Additionally, very small amounts of Sulfolobus spp. were detected. This study, along with other recent studies on the diversity of thermoacidophiles involved in the solubilization of copper from chalcopyrite concentrates, revealed that a wider variety of thermoacidophiles are involved in bioprocessing of metal sulfides, and showed that A. brierleyi should be considered an important biomining acidophile.