Thermophilic Culture Research Articles

Methyl-reducing methanogenesis by a thermophilic culture of Korarchaeia.

Methanogenesis mediated by archaea is the main source of methane, a strong greenhouse gas, and thus is critical for understanding Earth's climate dynamics. Recently, genes encoding diverse methanogenesis pathways have been discovered in metagenome-assembled genomes affiliated with several archaeal phyla1-7. However, all experimental studies on methanogens are at present restricted to cultured representatives of the Euryarchaeota. Here we show methanogenic growth by a member of the lineage Korarchaeia within the phylum Thermoproteota (TACK superphylum)5-7. Following enrichment cultivation of 'Candidatus Methanodesulfokora washburnenis' strain LCB3, we used measurements of metabolic activity and isotope tracer conversion to demonstrate methanol reduction to methane using hydrogen as an electron donor. Analysis of the archaeon's circular genome and transcriptome revealed unique modifications in the energy conservation pathways linked to methanogenesis, including enzyme complexes involved in hydrogen and sulfur metabolism. The cultivation and characterization of this new group of archaea is critical for a deeper evaluation of the diversity, physiology and biochemistry of methanogens.

Influence of thermophilic and mesophilic cultures blended with buffalo milk on camel milk cheese quality and proteolysis

SummaryCamel milk and its products are gaining higher demand due to their attractive nutritional and therapeutic profile. However, manufacturing of camel milk cheese (CMC) is a challenging task. This study aimed to compare the quality of camel milk cheese made using different starter cultures. Four groups CM (camel milk + mesophilic cultures), CT (camel milk + thermophilic cultures), CBM (camel milk + 10% buffalo milk using mesophilic cultures), and CBT (camel milk + 10% buffalo milk using thermophilic cultures) were created. The cheeses were stored for 60 days at 4 °C to assess their physicochemical, sensory, and proteolytic properties. Results indicated highest moisture content in CM (70.19%) as opposed to the lowest moisture in CBT (55.19%). In addition, CM and CBM revealed lower acidity (0.63% and 0.66%) in contrast with CT and CBT (0.78% and 0.83%, respectively). On the other hand, CBM and CBT exhibited higher contents of protein (21.04, 21.57%) and fat (17.65, 17.70%) during storage. All quality indices of cheese samples significantly declined (P < 0.01) during storage excluding acidity. Sensory analyses revealed that panellists highly preferred CBT treatment followed by CBM and CT. Furthermore, urea‐PAGE and RP‐HPLC were performed for the proteolytic study of cheese samples. The study revealed that the presence of buffalo milk and thermophilic cultures in cheese resulted in a decrease concentration of intact caseins (αs1, β‐, and κ‐caseins) as storage days passed. The degree of proteolysis was found to be higher in CBT compared to CBM, CT, and CM. Based on the results, it was concluded that the use of pasteurisation at 65 °C for 30 min, pH of 5.5, 0.06% CaCl2, and the incorporation of 10% buffalo milk using thermophilic cultures (Lactobaccilus bulgaricus and Streptococcus thermophilus) resulted in desirable quality characteristics.

COMPARATIVE STUDY OF FERMENTATION PROCESSES OF YAK AND COW MILK

Yak milk is a valuable raw material for the production of functional food. Investigation of the fermentation process with commercial starter cultures is conducted to find suitable starters for producing fermented yak milk products on an industrial scale using modern rheological, microbiological, and sensorial methods. According to the acidification curve and rheological measurement, the gel-forming time of yak milk with mesophilic, thermophilic and mixed (meso-thermophilic) starter cultures was not significantly different from cow milk; but, yak milk had a significantly higher gel strength in all cases. The viable bacteria count of fermented samples with thermophilic starters was 8.23 log CFU∙mL-1 for cow milk and 9.12 log CFU∙mL-1 for yak milk. During cold storage (72 h), the CFU in yak milk remained higher (98.48% – 101.22%) during the storage period than that of cow milk (90.33% – 95.06%). The sensorial property scores of yak milk fermented with mixed starters were the highest, followed by those of milk fermented with mesophilic and thermophilic starter cultures. The obtained parameters show that mesophilic and mixed cultures are advantageous in terms of sensorial and probiotic properties, and thermophilic culture - in terms of gel strength. All this information is useful for the development of new dairy products.

Evaluation of the effects of L-cysteine addition on the bioleaching of zinc and cadmium from sphalerite flotation concentrate

The primary zinc ore in the world is sphalerite, and roasting-leaching-electrowinning (RLE) is the most typical method of processing. This method has different disadvantages, and bioleaching is an alternative and eco-friendly method for sphalerite treatment. The major drawback of bioleaching is its slow kinetics. In this study, an evaluation of the effects of L-cysteine addition on the bioleaching of zinc and cadmium from a concentrate of sphalerite was investigated in the presence of mixed cultures of mesophilic and moderately thermophilic microorganisms. The results showed that L-cysteine addition increased the dissolution rates of zinc and cadmium while reducing them at higher concentrations. The optimal conditions were achieved by using 0.1 g/L of L-cysteine in mesophilic cultures with zinc recovery of 73.92% and cadmium recovery of 81.45%, respectively. Similarly, zinc recovery was 92.54% in thermophilic cultures, while cadmium recovery was 88.66%. Field emission scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) analyses have detected the formation of jarosite and elemental sulfur on sphalerite surfaces in mesophilic conditions, as well as ammoniojarosite and elemental sulfur in moderately thermophilic conditions. Fourier transform infrared (FTIR) spectroscopy was employed to identify molecular structures and functional groups on the surface of bioleached residues in mesophilic and moderately thermophilic conditions. To assess the environmental risk of residues, a combination of synthetic precipitation leaching procedure (SPLP) and toxicology characteristic leaching procedure (TCLP) methods were used.

Optimizing biohydrogen production yields by employing locally isolated thermophilic bacteria from hot springs

A climate-neutral economy is anticipated to rely heavily on hydrogen because it enables emission-free transportation, heating, and manufacturing operations. Biohydrogen can be produced from various kinds of biological waste making the interest high. However, the yield and efficiency of the processes are still challenging. This study applied Box-Behnken statistical experimental design to investigate the influence of temperature (oC), pH, and CO volume (mL) together with the amount of Fe+2, Zn+2, and Ni+2 to enhance biohydrogen production yields from thermophilic cultures, both mixed and pure cultures isolated from hot springs in Izmir, Türkiye. The maximum H2 yields were reported as 0.13 mmolH2/mmolCO for mixed cultures, and the pure culture reached 2.5 fold higher yield (0.44 mmolH2/mmolCO). Bench-scale bioreactor with a custom-design micro sparger was successfully run for 7 days (highest 0.25 mmolH2/mmol CO). This is the first report in the literature with local isolates to demonstrate the optimization of H2 yields with a comparative approach, and scale-up in a 2 L bench scale bioreactor. The viability of using novel thermophilic isolates as biohydrogen producers was successfully proven.

JUSTIFICATION OF THE SHELF LIFE OF FERMENTED DAIRY PRODUCTS BASED ON GOAT'S MILK

The article presents the results of studies on the number of survival of lactic acid bacteria, which determine the shelf life of fermented milk products during storage. The studies were carried out at the Department of Food Biotechnology of the Almaty Technological University, as well as in the laboratory of biotechnology of specialized products and dietary supplements of the Kazakh Academy of Nutrition. In the preparation of fermented milk products, a starter consisting of Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus fermentum 14 was used. Lactobacillus fermentum 14, as a probiotic, exhibits antagonistic properties, which helps to extend the shelf life and does not allow yeast and mold to grow in the final products. Lactobacillus bulgaricus, in turn, synthesizes a specific enzyme, peptidoglycan hydrolase, which is responsible for the hydrolysis of peptidoglycan which is an important component of the bacterial cell wall. When dairy products are enriched with biologically active substances of berry syrups Lactobacillus bulgaricus actively synthesizes extracellular polysaccharides, which improve the structure of the clot, increase stability and prevent syneresis. Streptococcus thermophilus is a mild curing thermophilic culture with a high degree of viscosity that promotes the formation of a good clot. Rosehip, hawthorn and mountain ash syrups, grape peel extract enriched fermented milk products with biologically active substances, which improved the growth and development of lactic acid microorganisms. The addition of phytofillers not only enhance the microscopic picture, but also improves the consistency and odors of the products.

Optimization of spray dried yogurt and its application to prepare functional cookies.

Spray-dried yogurt powder (SDYP) has shelf stability and other functional properties that improve solubility and facilitate the use, processing, packaging, and transportation of other food derivatives, such as bread and pastries on a large scale. The present research was conducted to develop SDYP and further its utilization to prepare functional cookies. Yogurt was spray-dried by employing different outlet air temperatures (OAT) (65°C, 70°C & 75°C) and inlet air temperature (IAT) (150°C, 155°C & 160°C). Spray drying shows that increasing the temperature increases nutritional loss, whereas S. thermophilus culture shows resistance to the intensive heat approaches. On the other hand L. delbrueckii subsp. Bulgaricus culture was found to be significantly affected. A total of 4 treatments, including one control for the functional cookies development. A directly proportional relation was investigated between the increasing concentration of SDYP and baking characteristics and cookie's mineral and protein profile. Bioactive parameters like antioxidant activity of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and total phenolic content (TPC) were also affected significantly. The sensory profile shows an incline towards T0 (0% SDYP) to T3 (10% SDYP) in all attributes but starts to decline when the concentration of SDYP reaches 15%. This study suggests that by employing a certain combination of temperatures (OAT: 60°C IAT: 150°C); maximum survival of inoculated culture can be achieved, and this powder can be utilized in the development of functional cookies with enhanced sensory as well as biochemical characteristics significantly (P< 0.05).

Bioleaching of zinc, copper and antimony from a tetrahedrite concentrate using acidophilic microorganisms

In this study, bacterial leaching of zinc, copper and antimony from tetrahedrite concentrate was investigated with mixed mesophilic and mixed moderately thermophilic microorganisms. The mineralogical studies showed that tetrahedrite ((Cu,Fe)12Sb4S13), ankerite (Ca(Fe,Mg)(CO3)2) and quartz (SiO2) are the main mineral phases. During bioleaching experiments, the effects of different chemicals and factors such as graphite, silver nitrate, L-cysteine, pyrite, and mechanical activation were studied for the first time. It was found that the removal of toxic antimony from tetrahedrite concentrate by alkaline Na2S-NaOH facilitates the growth of microorganisms and extraction rates of metals. The maximum extraction of zinc, copper and antimony was 99.7%, >99.9% and 92.8% with ORP, cell counts and Fe(III) concentration of 666 mV, 108 cells/ml and 9.58 g/l, respectively, using the moderately thermophilic culture with the tetrahedrite residue obtained after alkaline leaching (low-Sb). Under the same control leaching conditions with H2SO4, the maximum extraction of zinc, copper and antimony was 26.2%, 46.1% and 10.7% with low ORP and Fe(III) concentration of 464 mV and 5.18 g/l, respectively. The results showed that bacterial leaching of tetrahedrite concentrate significantly improves the extraction of metals compared to control test. As an important result, mechanical activation by planetary mill played a significant role during tetrahedrite bioleaching. Residual analysis proved the formation of antimony sulfide (Sb2S5) and jarosite (K3Fe9S6O42H18) phase as dissolution inhibitors and acanthite phase (Ag2S) as accelerator for dissolution kinetics.

Storage related changes in short-set cream cheese manufactured using thermophilicstarter culture and direct acidification technique

Cream cheese is a fresh acid curd variety with semi-soft body, manufactured by the gradual, quiescent acid gelation of milk. The typical flavour profile of cream cheese is cultured diacetyl with little bit lactic acid flavour as well as aroma. It is envisaged that demand of cream cheese will increase in upcoming years due to its application as spread and it having higher nutritional value compared to similar products available in the market. The purpose of the study was to observe changes in sensory, physicochemical and microbial quality during storage in short-set cream cheese samples manufactured by using thermophilic starter culture and direct acidification technique and was compared with control cream cheese manufactured using mesophilic starter culture. All the three samples i.e., control cream cheese made using mesophilic culture (MC), cream cheese made using thermophilic culture (TC) and cream cheese made using laboratory grade lactic acid (SLA) were stored in re-closable polypropylene tubs at 7 ± 2°C. During storage study, flavour score, FFA, tyrosine value were significantly affected by storage period (P), treatment (T) and the interaction of T × P. Moisture, pH, titratable acidity, sensory parameters (except flavour) were significantly affected only by storage period (P) and treatment(T). All the three samples had storage stability up to 15 days at 7± 2°C.

Green extraction of nickel and valuable metals from pyrrhotite samples with different crystallographic structures through acidophilic bioleaching.

Nowadays, due to the strategic status of nickel in the global market, utilizing its disregarded resources like low-grade nickel containing pyrrhotite is of significant importance. A comprehensive set of experiments and analyses were performed to determine the bioleaching capability and mechanism for nickel extraction from hexagonal and monoclinic pyrrhotite. Over 95% Ni extraction was achieved from the hexagonal pyrrhotite sample. Ni extraction from the monoclinic sample reached its maximum value of 67% and 90% at 3% pulp density, with mixed mesophilic and moderately thermophilic cultures, respectively. Characterization analyses indicated that jarosite and elemental sulfur formation in mixed mesophilic bioleaching reduced the samples' bio-oxidation rate and metal dissolution. The kinetics study revealed that the controlling step in thermophilic bioleaching is the chemical reaction; however, the mixed control model was best fitted on mesophilic data. Electrochemistry studies confirmed bioleaching results and indicated that monoclinic pyrrhotite's oxidation rate under the operating conditions is faster than hexagonal pyrrhotite, and the temperature positively correlates with the oxidation rate. Toxicity assessment analysis showed that the final residues of both bioleached samples could be considered environmentally safe.

ANTIMICROBIAL, ANTIBIOFILM, AND PROBIOFILM EFFECTS OF GALLIC ACID ON EXOPOLYSACCHARIDE-DEPENDENT AND -INDEPENDENT BIOFILM OF MODEL STRAINS STREPTOCOCCUS THERMOPHILUS CNRZ 447 AND STAPHYLOCOCCUS AUREUS ATCC 43300

This study aimed to identify an approach that can simultaneously prevent biofilm-associated damage from Staphylococcus aureus, the most prevalent agent of mastitis in cattle in Algeria and dairy product spoiler, and improve the properties of dairy starters and probiotics. Selected strains Streptococcus thermophilus CNRZ 447 and methicillin-resistant S. aureus ATCC 43300 were cultured independently in broth containing increasing concentrations of gallic acid. S. aureus ATCC 43300 was shown to be an exopolysaccharide-independent biofilm producer. Gallic acid exhibited a dose- and strain-dependent antimicrobial, antibiofilm, and probiofilm effects. The molecule of gallic acid improved the exopolysaccharide production ability of S. thermophilus and altered adhesion determinants (e.i., auto-aggregation and surface hydrophobicity) of both strains. This study indicated that biofilm-associated phenotypic characteristics, such as the composition of the extracellular structure, should be determinant in the action of gallic acid on microorganisms. The intriguing stimulation of S. aureus ATCC 43300 biofilm by gallic acid could not allow us to conclude on its use as a biofilm preventing compound. Nonetheless, gallic acid was demonstrated to be a potent antimicrobial compound against S. aureus and an enhancer of biofilm phenotypic characteristics (including adhesion properties and exopolysaccharide production) of S. thermophilus. These observations suggest that gallic acid or related phenolic compounds can be used to improve the properties of probiotics and the performance of microbial cell factories. Subsequently, gallic-acid adapted S. thermophilus cultures could be explored to disperse S. aureus in dairy products and industrial materials.

Low-Cost Clamp-On Photometers (ClampOD) and Tube Photometers (TubeOD) for Online Cell Density Determination.

Optical density (OD) measurement is the gold standard to estimate microbial cell density in aqueous systems. Recording microbial growth curves is essential to assess substrate utilization, gauge sensitivity to inhibitors or toxins, or determine the perfect sampling point. Manual sampling for cuvette-photometer-based measurements can cause disturbances and impact growth, especially for strictly anaerobic or thermophilic microbes. For slow growing microbes, manual sampling can cause data gaps that complicate analysis. Online OD measurement systems provide a solution, but are often expensive and ill-suited for applications such as monitoring microbial growth in custom or larger anaerobic vessels. Furthermore, growth measurements of thermophilic cultures are limited by the heat sensitivity of complex electronics. Here, we present two simple, low-cost, self-assembled photometers—a “TubeOD” for online measurement of anaerobic and thermophilic cultures in Hungate tubes and a “ClampOD” that can be attached to virtually any transparent growth vessel. Both OD-meters can be calibrated in minutes. We detail the manufacturing and calibration procedure and demonstrate continuous acquisition of high quality cell density data of a variety of microbes, including strict anaerobes, a thermophile, and gas-utilizing strains in various glassware. When calibrated and operated within their detection limits (ca. 0.3–90% of the photosensor voltage range), these self-build OD-meters can be used for continuous measurement of microbial growth in a variety of applications, thereby, simplifying and enhancing everyday lab operations.

Compositional and Functional Characteristics of Feta-Type Cheese Made from Micellar Casein Concentrate.

Micellar casein concentrate (MCC) is a high protein ingredient (obtained by microfiltration of skim milk) with an elevated level of casein as a percentage of total protein (TP) compared to skim milk. It can be used as an ingredient in cheese making. Feta-type cheese is a brined soft cheese with a salty taste and acid flavor. We theorize that Feta-type cheese can be produced from MCC instead of milk, which can improve the efficiency of manufacture and allow for the removal of whey proteins before manufacturing Feta-type cheese. The objectives of this study were to develop a process of producing Feta-type cheese from MCC and to determine the optimum protein content in MCC to make Feta-type cheese. MCC solutions with 3% (MCC-3), 6% (MCC-6), and 9% (MCC-9) protein were prepared and standardized by mixing water, MCC powder, milk permeate, and cream to produce a solution with 14.7% total solids (TS) and 3.3% fat. Thermophilic cultures were added at a rate of 0.4% to MCC solutions and incubated at 35 °C for 3 h to get a pH of 6.1. Subsequently, calcium chloride and rennet were added to set the curd in 20 min at 35 °C. The curd was then cut into cubes, drained for 20 h followed by brining in 23% sodium chloride solutions for 24 h. Compositional analysis of MCC solutions and cheese was carried out. The yield, color, textural, and rheological measurements of Feta-type cheese were evaluated. Feta-type cheese was also made from whole milk as a control. This experiment was repeated three times. The yield and adjusted yield of Feta-type cheese increased from 19.0 to 54.8 and 21.4 to 56.5, respectively, with increasing the protein content in MCC from 3% to 9%. However, increasing the protein content in MCC did not show significant differences in the hardness (9.2–9.7 kg) of Feta-type cheese. The color of Feta-type cheese was less white with increasing the protein content in MCC. While the yellowish and greenish colors were high in Feta-type cheese made from MCC with 3% and 6% protein, no visible differences were found in the overall cheese color. The rheological characteristics were improved in Feta-type cheese made from MCC with 6% protein. We conclude that MCC with different levels of protein can be utilized in the manufacture of Feta-type cheese.

Bioleaching of cobalt from magnetite-rich cobaltite-bearing ore

Cobaltite (CoAsS) is a strategically important mineral. This work investigated the bioleaching of cobaltite-bearing magnetite-rich ore. The bioleaching of cobaltite was carried out with mixed mesophilic and moderately thermophilic cultures at 4, 8, and 12% pulp densities in shake flasks. The bioleaching with mesophiles at 8% pulp dissolved up to 89% of the cobalt content, higher than the bioleaching of 4% pulp and abiotic control test. Additional Fe3+ in the leach solution increased cobalt dissolution in the control test. The bioleaching by moderate thermophiles reached 97% cobalt dissolution in 16 d contact time. Kinetic modeling indicated that the controlling step of bioleaching with moderate thermophiles was the chemical reaction, while the diffusion was the rate-limiting factor for the bioleaching with mesophiles.

Formulation and evaluation of whey based ready-to-serve therapeutic beverage from Aloe debrana juice

The research was conducted with the aim to formulate and evaluate whey-based ready-toserve (RTS) therapeutic beverage from Aloe debrana juice and determine the phytochemical composition and sensory acceptability of the formulated therapeutic beverage. The whey and Aloe debrana juice were prepared in three blending ratios including WA1 (70:30), WA2 (80:20), and WA3 (90:10) respectively; and control (100% whey) with the addition of ingredients such as: sugar 15 g/100 mL, vanilla 0.1 g/100 mL, starter culture of Streptococcus thermophiles and Lactobacillus bulgaricus (0.02 g/100 mL). The four formulations were generated by Central composite design (CCD). The formulated raw materials range 70% to 90% whey, 10% to 30% Aloe debrana juice, and control (100% whey). Design expert 7.0 software was used to formulate the two components. Therapeutic beverages were fermented for 9 hrs at 37°C and 120 rpm using shaker incubator. Mineral contents (ppm) of the formulated beverage ranged from 375 to 573.01, 1244.02 to 1491.03, 460.01 to 742, and 93.02 to 107.0 of sodium, potassium, calcium, and magnesium, respectively. Phytochemical composition (mg/100 g) of the formulated beverages and Aloe debrana juice ranged from 43.20 to 147.56, and 4.98 to 11.17 for phytic acid and tannin respectively. The phytic acid and tannin content of Aloe debrana juice was (467.6 mg/100 g) and (130.1 mg/100 g), respectively. The therapeutic beverages were stored at room temperature for nine days without any change in sensory attributes. Sensory quality evaluation results showed that sample containing 70% whey and 30% Aloe debrana juice (WA1) scored the highest in overall acceptability 4.15, 3.7, 3.2, and 3.03 for the first, third, sixth, and ninth days of room storage temperature, respectively. Based on sensory quality evaluation parameters, therapeutic beverage containing 70% Whey and 30% Aloe debrana juice blend ratio revealed sensory acceptable for therapeutic beverage production. Therefore, it can be concluded that Aloe debrana juice can be incorporated with whey up to 30% to produce organolepticaly acceptable therapeutic beverages.

Pressure and Temperature Combined With Microbial Supernatant Effectively Inactivate Bacillus subtilis Spores.

Spores from the Bacillus species pose a challenge to the food industry because of their ubiquitous nature and extreme resistance. Accumulated evidence indicates that it is effective to induce spore germination homogenously before killing them. However, it is difficult to obtain and apply exogenous germination factors, which will affect food composition. Therefore, this study screened endogenous germinants from microorganisms by assessing the effect of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Lactiplantibacillus plantarum, and Streptococcus thermophilus cultures (cell-free) on B. subtilis spore germination. The results showed that the supernatants from these five microorganisms induced spore germination instead of sediments. Moreover, the supernatants of E. coli, B. subtilis, and S. cerevisiae exhibited higher germination rates than L. plantarum and S. thermophilus, and the induction effects were concentration-dependent. Furthermore, plate counting confirmed that the microbial supernatants induced the lowest spore germination ratio on strains B. subtilis FB85 [germination receptors (GRs) mutant] but not strains B. subtilis PB705 (PrkC mutant). In addition, B. subtilis and S. cerevisiae supernatants, combined with pressure and temperature, were effective in spore inactivation. The findings suggested that microbial supernatants may include agents that induce spore germination and may be used for spore inactivation.

Influence of co-substrates on anaerobic thermophilic degradation of syringaldehyde

Phenolic aldehydes resulting from lignocellulosic hydrolysis have often been considered harmful to the anaerobic cultures. Depending on the type of lignin, syringaldehyde present in hardwood and grass lignin can be produced as a by-product of its breakdown in varying extents. The influence of two mixed co-substrates, glucose and sodium acetate, on syringaldehyde degradation by anaerobic thermophilic cultures (50±2 °C) was assessed in the present work. Based on the monitoring of pH, chemical oxygen demand (COD) levels and UV–visible spectra in samples collected from batch bioreactors, it can be concluded that syringaldehyde is more prone to undergo anaerobic degradation in the presence of co-substrates. Both a reduction of the acclimatization period and an improvement in the COD removal (92.0%) were observed. As the only carbon source, syringaldehyde seems to be hardly degraded rendering a low efficiency of COD removal (53.2%). Some insights on the main alterations of syringaldehyde by anaerobic thermophilic cultures are also given.

Adaptation and characterization of thermophilic anammox in bioreactors

Anammox, the oxidation of ammonium with nitrite, is a key microbial process in the nitrogen cycle. Under mesophilic conditions (below 40 °C), it is widely implemented to remove nitrogen from wastewaters lacking organic carbon. Despite evidence of the presence of anammox bacteria in high-temperature environments, reports on the cultivation of thermophilic anammox bacteria are limited to a short-term experiment of 2 weeks. This study showcases the adaptation of a mesophilic inoculum to thermophilic conditions, and its characterization. First, an attached growth technology was chosen to obtain the process. In an anoxic fixed-bed biofilm bioreactor (FBBR), a slow linear temperature increase from 38 to over 48 °C (0.05–0.07 °C d−1) was imposed to the community over 220 days, after which the reactor was operated at 48 °C for over 200 days. Maximum total nitrogen removal rates reached up to 0.62 g N L−1 d−1. Given this promising performance, a suspended growth system was tested. The obtained enrichment culture served as inoculum for membrane bioreactors (MBR) operated at 50 °C, reaching a maximum total nitrogen removal rate of 1.7 g N L−1 d−1 after 35 days. The biomass in the MBR had a maximum specific anammox activity of 1.1 ± 0.1 g NH4+-N g−1 VSS d−1, and the growth rate was estimated at 0.075–0.19 d−1. The thermophilic cultures displayed nitrogen stoichiometry ratios typical for mesophilic anammox: 0.93–1.42 g NO2--Nremoved g−1 NH4+-Nremoved and 0.16–0.35 g NO3--Nproduced g−1 NH4+-Nremoved. Amplicon and Sanger sequencing of the 16S rRNA genes revealed a disappearance of the original “Ca. Brocadia” and “Ca. Jettenia” taxa, yielding Planctomycetes members with only 94–95% similarity to “Ca. Brocadia anammoxidans” and “Ca. B. caroliniensis”, accounting for 45% of the bacterial FBBR community. The long-term operation of thermophilic anammox reactors and snapshot views on the nitrogen stoichiometry, kinetics and microbial community open up the development path of thermophilic partial nitritation/anammox. A first economic assessment highlighted that treatment of sludge reject water from thermophilic anaerobic digestion of sewage sludge may become attractive.

Safety and technological application of autochthonous Streptococcus thermophilus cultures in the buffalo Mozzarella cheese

Thermophilic and mesophilic lactic acid bacteria (LAB), such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, and Lactococcus lactis, play a crucial role in the technological and sensory quality of Mozzarella cheese. In this study, the safety (genes encoding virulence factors and antibiotic resistance) and acidifying activity of autochthonous S. thermophilus cultures were evaluated in order to choose the most suitable strain for industrial application. The safe and good acidifying culture was tested in two buffalo Mozzarella cheese batches: Mozzarella cheeses produced with autochthonous culture (SJRP107) and commercial culture (STM5). The cultivable LAB was evaluated by culture-dependent method (plate counting) and the quantification of S. thermophilus cultures (commercial and autochthonous) were evaluated by culture-independent method RealT-qPCR (real-time quantitative polymerase chain reaction). The texture, physicochemical and proteolytic properties of the Mozzarella cheeses were similar for both batches. The nonstarter LAB count was higher during manufacture than in the storage, and the RealT-qPCR indicated the presence of S. thermophilus culture until the end of storage. S. thermophilus SJRP107 presented high potential for safety application in the production of Mozzarella cheese. Furthermore, considering the culture characteristics and their relationship with product quality, further studies could be helpful to determine their effect on the sensory characteristics of the cheese.

Changes in Proteolysis in Fermented Milk Produced by Streptococcus thermophilus in Co-Culture with Lactobacillus plantarum or Bifidobacterium animalis subsp. lactis During Refrigerated Storage.

Proteolysis in fermented milk, a complex and dynamic process, depends on the starter cultures used. This study aimed to evaluate the influence of Lactobacillus plantarum or Bifidobacterium animalis subsp. lactis, or both, co-fermented with Streptococcus thermophilus, on the changes in the proteolysis profile of fermented milk during 21-day storage at 4 °C, including the pH value, proteolytic degree, protease activity, aminopeptidase activity, free amino acid content, and electrophoresis performance. The results showed that the treatments with co-cultures exhibited a higher amount of free amino groups and neutral protease activity at an extracellular level, whereas lower pH values and aminopeptidase activities towards the six substrates at an intracellular level than the ones with a single-strain of S. thermophilus over the refrigerated storage were observed. In co-fermentation with S. thermophilus, B. animalis subsp. lactis did not significantly affect the concentrations of most free amino acids, while contributions of L. plantarum were found. Electrophoresis indicated that the mixed starters, especially the co-cultures containing L. plantarum, showed a stronger degradation for caseins than the pure S. thermophilus culture. These findings suggest that culture combinations may influence the proteolysis characteristics of the fermented products, and probiotic cultures must be carefully chosen for fermented production.