Lipase-catalysed kinetic resolution of racemates at temperatures from 40 °C to 160 °C in supercritical CO2

The reaction rate and selectivity of the enzymatic kinetic resolution of ibuprofen and 1-phenylethanol with supercritical CO2 as solvent were studied in a batch reactor from 40 °C to 160 °C. The commercial enzyme, Novozym 435, remained partly active for at least 14 h up to 140 °C at 15 MPa. The maximum reaction rate for the esterification of 1-phenylethanol and ibuprofen was at about 90 °C. The enantiomeric excess for 1-phenylethanol exceeds 99% and was temperature independent. Selectivity for ibuprofen esterification reached a lower enantiomeric excess of 61% caused by equilibrium adjustment. The results show that with supercritical CO2 as reaction medium enzymes remain active above 100 °C.     

Chemotactic behavior of Campylobacter spp. in function of different temperatures (37 °C and 42 °C)

The chemotactic behaviour of Campylobacter strains was determined in the presence of different amino acids at two temperatures (37 °C and 42 °C). Two strains of catalase positive (Campylobacter jejuni) and negative (Campylobacter sputurum) Campylobacter were isolated from river water in Tonekabon, Iran and identified by phenotyping and 16srRNA Gene sequencing methods. Chemotactic responses of the isolates were assessed toward a variety of amino acids viz., L-cystine, L-asparagine, L-histidine, L-aspartic acid, L-serine, L-phenylalanine, L-leucine and L-tryptophan by disc and capillary methods at two temperatures: 37 °C and 42 °C. C. jejuni showed positive chemotactic response towards L-cystine,L-tryptophan, L-phenylalanine, - L-leucine, L-asparagine and L-Serine at both, 37 °C and 42 °C however, it was greater at 37 °C. C. sputurum showed negative or weak response towards all of the amino acids. In addition, C. jejuni illustrated strong chemotactic response to L-asparagine follow by L-serine and weak chemotaxis response to L-phenylalanine and L-cysteine at 37 °C. Overall, C. jejuni showed relatively strong chemotactic response to some amino acids, likewise it was greater at 37 °C. Hence, the human body temperature (37 °C) in compared to avian body temperature (42 °C) probably promotes chemotactic response of C. jejuni, which it might be a reason for causing disease in human being compared to avian.     

Long-term,large scale cryopreservation of insect cells at −80 °C

Standard tissue culture methods advise freezing cells in small aliquots (≤1 × 10⁷ cells in 1 mL), and storing in liquid nitrogen. This is inconvenient for laboratories culturing large quantities of insect cells for recombinant baculovirus expression, owing to the length of time taken to produce large scale cultures from small aliquots of cells. Liquid nitrogen storage requires use of specialized cryovials, personal protective equipment and oxygen monitoring systems. This paper describes the long-term, large scale cryopreservation of 8 × 10⁸ insect cells at −80 °C, using standard 50 mL conical tubes to contain a 40 mL cell suspension. Sf9, Sf21 and High 5 cells were recovered with a viability > 90 % after storage for one year under these conditions, which compared favorably with the viability of cells stored in liquid nitrogen for the same length of time. Addition of green fluorescent protein encoding baculovirus demonstrated that cells were “expression ready” immediately post thaw. Our method enables large scale cultures to be recovered rapidly from stocks cryopreserved at −80 °C, thus avoiding the inconvenience, hazards and expense associated with liquid nitrogen.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-014-9781-5) contains supplementary material, which is available to authorized users.     

Performance and microbial community composition dynamics of aerobic granular sludge from sequencing batch bubble column reactors operated at 20 °C, 30 °C, and 35 °C

Two bubble column sequencing batch reactors fed with an artificial wastewater were operated at 20 °C, 30 °C, and 35 °C. In a first stage, stable granules were obtained at 20 °C, whereas fluffy structures were observed at 30 °C. Molecular analysis revealed high abundance of the operational taxonomic unit 208 (OTU 208) affiliating with filamentous bacteria Leptothrix spp. at 30 °C, an OTU much less abundant at 20 °C. The granular sludge obtained at 20 °C was used for the second stage during which one reactor was maintained at 20 °C and the second operated at 30 °C and 35 °C after prior gradual increase of temperature. Aerobic granular sludge with similar physical properties developed in both reactors but it had different nutrient elimination performances and microbial communities. At 20 °C, acetate was consumed during anaerobic feeding, and biological phosphorous removal was observed when Rhodocyclaceae-affiliating OTU 214 was present. At 30 °C and 35 °C, acetate was mainly consumed during aeration and phosphorous removal was insignificant. OTU 214 was almost absent but the Gammaproteobacteria-affiliating OTU 239 was more abundant than at 20 °C. Aerobic granular sludge at all temperatures contained abundantly the OTUs 224 and 289 affiliating with Sphingomonadaceae indicating that this bacterial family played an important role in maintaining stable granular structures.     

Cryopreservation at −20 and −70 °C of Pleurotus ostreatus on Grains

Alternative substrates for cryopreservation at −20 °C have been little explored for basidiomycetes and could bring new possibilities of lower cost cryopreservation. Nevertheless, freezing temperatures between −15 and −60 °C are very challenging because they frequently result in cryoinjuries. The objective of this study was to evaluate substrates associated to cryoprotective agents for Pleurotus ostreatus cryopreservation at −20 or −70 °C in order to develop alternative techniques for basidiomycete cryopreservation. P. ostreatus was grown on potato dextrose agar or whole grains of oat, wheat, rice or millet and transferred to cryovials with cryoprotective solution with 1 % dimethyl sulfoxide, 5 % glycerol, 10 % saccharose, 4 % glucose, 6 % polyethylene glycol-6000 or 5 % malt extract. The mycelium in the cryovials were cryopreserved at −20 or −70 °C and recovered for evaluation of the mycelial growth viability after 1 and 3 years. Both substrates and cryoprotectants affect the viability of the mycelial growth cryopreserved at −20 or −70 °C; wheat grains combined with cryoprotectants such as saccharose or glucose are effective for keeping mycelium viable after cryopreservation at −20 °C for 1 or 3 years; for cryopreservation at −70 °C after 1 or 3 years, any substrate combined with any cryoprotectant is effective for preserving the mycelium viable, except for millet grains with polyethylene glycol after 3 years; semi-permeable cryoprotective agents such as saccharose and glucose are the most effective for cryopreservation at −20 or −70 °C for at least 3 years.     

Cartilage storage at 4 °C with regular culture medium replacement benefits chondrocyte viability of osteochondral grafts in vitro

Maintenance of articular cartilage allografts in culture media is a common method of tissue storage; however, the technical parameters of graft storage remain controversial. In this study, we examined the optimal temperature and culture medium exchange rate for the storage of osteochondral allografts in vitro. Cylindrical osteochondral grafts (n = 120), harvested from the talar joint surface of ten Boer goats, were randomly classified into four groups and stored under the following conditions: Group A1 was maintained at 4 °C in culture medium that was refreshed every 2 days; Group A2 was maintained at 4 °C in the same culture medium, without refreshing; Group B1, was maintained at 37 °C in culture medium that was refreshed every 2 days; Group B2, was maintained at 37 °C in the same culture medium, without refreshing. Chondrocyte viability in the grafts was determined by ethidium bromide/fluorescein diacetate staining on days 7, 21, and 35. Proteoglycan content was measured by Safranin-O staining. Group A1 exhibited the highest chondrocyte survival rates of 90.88 %, 88.31 % and 78.69 % on days 7, 21, and 35, respectively. Safranin O staining revealed no significant differences between groups on days 21 and 35. These results suggest that storage of osteochondral grafts at 4 °C with regular culture medium replacement should be highly suitable for clinical application.     

Population growth and allergen accumulation of Dermatophagoides farinae cultured at 20 and 25 °C

House dust mites are cultured to obtain mite allergen material to produce allergen extracts (vaccines) for diagnostic tests, immunotherapy, and research purposes. Research laboratories and manufacturers have their own culturing protocols to grow these mites and these may vary between manufacturers and between research laboratories. The temperature at which mites are cultured may influence the allergen composition, allergen ratio of Der 1: Der 2 and endotoxin levels in the extracts produced from these cultured mites. In order to produce standardized and uniform extracts, across the industry and in various research laboratories, the influence of culture conditions must be understood. Here we determined how temperature affects mite population growth rates, dynamics of allergen production, Der f 1: Der f 2 ratio and endotoxin levels in extracts made from Dermatophagoides farinae mites cultured at 20 and 25 °C. We found that Der f 1 and Der f 2 accumulated exponentially in the cultures with Der f 1 accumulating faster than Der f 2. When the live mite populations peaked, the ratios for Der f 1: Der f 2 were 4.1 and 4.7 for cultures reared at 20 and 25 °C, respectively. Most of the Der f 1 and Der f 2 allergen in whole cultures is not in mite bodies and is lost when the mite material is washed. Thus, if the ratio of Der f 1 and Der f 2 is an important consideration for commercial and research extracts, then the temperature at which the mites are cultured and the collection procedure are important considerations.     

Activation of Bacillus spores at moderately elevated temperatures (30–33 °C)

The time/temperature profiles experienced by spores on the track from their natural sporulation environment to consumable food products may be highly diverse. Temperature has been documented as an important factor that may activate spores, i.e. potentiates spores to germinate. There is, however, limited knowledge about the relationship between the expected temperature history and the subsequent germination characteristics of bacterial spores. We show here that the germination rate of five different Bacillus spore populations, represented by strains of Bacillus cereus, Bacillus weihenstephanensis, Bacillus pumilus, Bacillus licheniformis and Bacillus subtilis could be increased following 1 week storage at moderately elevated temperatures, 30–33 °C, compared to spores stored at 3–8 °C. The results imply that spores contamination routes to foods, specifically the temperature history, could be highly relevant data in predictive modeling of food spoilage and safety. Activation at these moderately elevated temperatures may be a native form of spore activation in their natural habitats, knowledge that also could be useful in development of decontamination strategies for mildly heated foods.     

Effect of culture pH on recombinant antibody production by a new human cell line, F2N78, grown in suspension at 33.0 °C and 37.0 °C

The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0–7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.     

Human Semen Refrigeration at + 4 °C: Bio-kinetic Characteristics

The aim of our study was to evaluate the bio-kinetic characteristics of human semen refrigerated for different periods and to compare the effects of refrigeration at +4 °C against cryopreservation of human sperm at −196 °C. Semen was obtained from 30 male partners of infertile couples (infertile subjects) with the following semen profile: sperm count ≥10 × 10⁶/ml; progressive motility ≥20%; atypical forms <70% and white blood cells <1.0 × 10⁶/ml. Fifteen normospermic subjects were also selected as controls (control subjects). The following tests were carried out on basal, refrigerated and cryopreserved sperm: a) sperm kinetic properties (by Superimposed Image Analysis System); b) the Hypoosmotic Viability Test (HVT) (combined Hypoosmotic Swelling and Viability Test). The results of the study showed that the percentage recovery of kinetic properties and of HVT were optimum for up to 48 h. After refrigeration for 72 h, a drastic decrease in straight motility recovery was observed. No significant differences were observed between cryopreservation and refrigeration at +4 °C for 48 h for motility or HVT recoveries in samples from control subjects. However, in infertile subjects, a significant decrease in straight progressive motility and HVT recoveries was observed in cryopreserved samples compared to those refrigerated for 48 h. Neither refrigeration nor cryopreservation led to the growth of pathogenic bacteria in any of the cases studied. Based on the above results, refrigeration could represent a useful alternative to the cryopreservation method.     

Effect of preservation of human semen sample at 4–6 and 25 °C on sperm motility

Sperm motility is the result of transverse movements that exist along its tail. It plays an important role in male fertility. The aim of this study was to evaluate the influence of keeping washed normozoospermic semen samples at 4–6 and 25 °C on the motility of spermatozoa. 26 semen samples of normozoospermic were washed twice in modified Ham’s F10 medium. Then, thirteen of the semen samples were kept in refrigerator (4–6 °C) and the remaining samples were stored in incubator (25 °C) for 12 days. On the 0 (immediately after sampling as control group), 1st, 2nd, 5th, 7th and the 12th days, the percentage of fast progressive (grade a), slow progressive (grade b), non-progressive (grade c) and immotile (grade d) sperm cells were calculated for each temperature. The data obtained from this study showed that the percentages of a, b and c grades of motile spermatozoa were significantly decreased (p?<?0.001) during 12 days at the both temperatures but reduction of these percentages has a gentle slope at 4–6 °C. There was no motile sperm after 12 days of storage. This study suggests that motile spermatozoa could be retrieved up to 7 days after the storage of washed normozoospermic men semen samples at 4–6 and 25 °C. Also, there were no motile sperm cells 12 days after sampling.     

Bioelectrochemical analysis of a hyperthermophilic microbial fuel cell generating electricity at temperatures above 80 °C

We examined whether a hyperthermophilic microbial fuel cell (MFC) would be technically feasible. Two-chamber MFC reactors were inoculated with subsurface microorganisms indigenous to formation water from a petroleum reservoir and were started up at operating temperature 80 °C. The MFC generated a maximum current of 1.3 mA 45 h after the inoculation. Performance of the MFC improved with an increase in the operating temperature; the best performance was achieved at 95 °C with the maximum power density of 165 mWm^?2, which was approximately fourfold higher than that at 75 °C. Thus, to our knowledge, our study is the first to demonstrate generation of electricity in a hyperthermophilic MFC (operating temperature as high as 95 °C). Scanning electron microscopy showed that filamentous microbial cells were attached on the anode surface. The anodic microbial consortium showed limited phylogenetic diversity and primarily consisted of hyperthermophilic bacteria closely related to Caldanaerobacter subterraneus and Thermodesulfobacterium commune.     

Ammonia oxidizing bacteria and archaea in horizontal flow biofilm reactors treating ammonia-contaminated air at 10 °C

The objective of this study was to demonstrate the feasibility of novel, Horizontal Flow Biofilm Reactor (HFBR) technology for the treatment of ammonia (NH₃)-contaminated airstreams. Three laboratory-scale HFBRs were used for remediation of an NH₃-containing airstream at 10 °C during a 90-d trial to test the efficacy of low-temperature treatment. Average ammonia removal efficiencies of 99.7 % were achieved at maximum loading rates of 4.8 g NH₃ m³ h^?1. Biological nitrification of ammonia to nitrite (NO₂ ^?) and nitrate (NO₃ ^?) was mediated by nitrifying bacterial and archaeal biofilm populations. Ammonia-oxidising bacteria (AOB) were significantly more abundant than ammonia-oxidising archaea (AOA) vertically at each of seven sampling zones along the vertical HFBRs. Nitrosomonas and Nitrosospira, were the two most dominant bacterial genera detected in the HFBRs, while an uncultured archaeal clone dominated the AOA community. The bacterial community composition across the three HFBRs was highly conserved, although variations occurred between HFBR zones and were driven by physicochemical variables. The study demonstrates the feasibility of HFBRs for the treatment of ammonia-contaminated airstreams at low temperatures; identifies key nitrifying microorganisms driving the removal process; and provides insights for process optimisation and control. The findings are significant for industrial applications of gas oxidation technology in temperate climates.     

Purinergic inhibition of Na+,K+,Cl− cotransport in C11-MDCK cells: Role of stress-activated protein kinases

Previously, we observed that sustained activation of P2Y₁ leads to inhibition of Na⁺,K⁺,Cl⁻ cotransport (NKCC) in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney. This study examined the role of stress-activated protein kinases (SAPK) in NKCC inhibition triggered by purinergic receptors. Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38. Activation of these kinases also occurred in anisomycin-treated cells. Surprisingly, we observed that compounds SP600125 and SB202190, known as potent inhibitors of JNK and p38 in cell-free systems, activated rather than inhibited phosphorylation of the kinases in C11 cells. Importantly, similarly to ATP, all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC. Thus, our results suggest that activation of JNK and/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y₁ agonists.     

Biodegradation of Petroleum Hydrocarbons in Seawater at Low Temperatures (0–5 °C) and Bacterial Communities Associated with Degradation

In this study biodegradation of hydrocarbons in thin oil films was investigated in seawater at low temperatures, 0 and 5 °C. Heterotrophic (HM) or oil-degrading (ODM) microorganisms enriched at the two temperatures showed 16S rRNA sequence similarities to several bacteria of Arctic or Antarctic origin. Biodegradation experiments were conducted with a crude mineral oil immobilized as thin films on hydrophobic Fluortex adsorbents in nutrient-enriched or sterile seawater. Chemical and respirometric analysis of hydrocarbon depletion showed that naphthalene and other small aromatic hydrocarbons (HCs) were primarily biodegraded after dissolution to the water phase, while biodegradation of larger polyaromatic hydrocarbons (PAH) and C₁₀–C₃₆ n-alkanes, including n-hexadecane, was associated primarily with the oil films. Biodegradation of PAH and n-alkanes was significant at both 0 and 5°C, but was decreased for several compounds at the lower temperature. n-Hexadecane biodegradation at the two temperatures was comparable at the end of the experiments, but was delayed at 0°C. Investigations of bacterial communities in seawater and on adsorbents by PCR amplification of 16S rRNA gene fragments and DGGE analysis indicated that predominant bacteria in the seawater gradually adhered to the oil-coated adsorbents during biodegradation at both temperatures. Sequence analysis of most DGGE bands aligned to members of the phyla Proteobacteria (Gammaproteobacteria) or Bacteroidetes. Most sequences from experiments at 0°C revealed affiliations to members of Arctic or Antarctic consortia, while no such homology was detected for sequences from degradation experiment run at 5°C. In conclusion, marine microbial communities from cold seawater have potentials for oil film HC degradation at temperatures ≤5°C, and psychrotrophic or psychrophilic bacteria may play an important role during oil HC biodegradation in seawater close to freezing point.     

Pathways of methanol conversion in a thermophilic anaerobic (55 °C) sludge consortium

The pathway of methanol conversion by a thermophilic anaerobic consortium was elucidated by recording the fate of carbon in the presence and absence of bicarbonate and specific inhibitors. Results indicated that about 50% of methanol was directly converted to methane by the methylotrophic methanogens and 50% via the intermediates H₂/CO₂ and acetate. The deprivation of inorganic carbon species [(HCO₃^–+CO₂)] in a phosphate-buffered system reduced the rate of methanol conversion. This suggests that bicarbonate is required as an electron (H₂) sink and as a co-substrate for the efficient and complete removal of the chemical oxygen demand. Nuclear magnetic resonance spectroscopy was used to investigate the route of methanol conversion to acetate in bicarbonate-sufficient and bicarbonate-depleted environments. The proportions of [1,2-¹³C]acetate, [1-¹³C]acetate and [2-¹³C]acetate were determined. Methanol was preferentially incorporated into the methyl group of acetate, whereas HCO₃^– was the preferred source of the carboxyl group. A small amount of the added H¹³CO₃^– was reduced to form the methyl group of acetate and a small amount of the added ¹³CH₃OH was oxidised and found in the carboxyl group of acetate when ¹³CH₃OH was converted. The recovery of [¹³C]carboxyl groups in acetate from ¹³CH₃OH was enhanced in bicarbonate-deprived medium. The small amount of label incorporated in the carboxyl group of acetate when ¹³CH₃OH was converted in the presence of bromoethanesulfonic acid indicates that methanol can be oxidised to CO₂ prior to acetate formation. These results indicate that methanol is converted through a common pathway (acetyl-CoA), being on the one hand reduced to the methyl group of acetate and on the other hand oxidised to CO₂, with CO₂ being incorporated into the carboxyl group of acetate.     

A method to maintain mammalian cells for days alive at 4 °C

This paper describes a method for the temporary storage of cultured cells. Cells from recently completed cell monolayers were trypsinized and then centrifuged. After centrifugation, the supernatant and pellet were kept at 4 °C for one week. After storage, the supernatant was discarded, the cells were resuspended and used for seeding new flasks and for titration of virus. The cells not only remained viable, but also rapidly formed new monolayers and allowed immediate infection and growth of viruses. We conclude that this method can be a helpful asset to cell culture experiments.     

One-stage partial nitritation/anammox at 15 °C on pretreated sewage: feasibility demonstration at lab-scale

Energy-positive sewage treatment can be achieved by implementation of oxygen-limited autotrophic nitrification/denitrification (OLAND) in the main water line, as the latter does not require organic carbon and therefore allows maximum energy recovery through anaerobic digestion of organics. To test the feasibility of mainstream OLAND, the effect of a gradual temperature decrease from 29 to 15 °C and a chemical oxygen demand (COD)/N increase from 0 to 2 was tested in an OLAND rotating biological contactor operating at 55–60 mg NH₄ ⁺–N?L^?1 and a hydraulic retention time of 1 h. Moreover, the effect of the operational conditions and feeding strategies on the reactor cycle balances, including NO and N₂O emissions were studied in detail. This study showed for the first time that total nitrogen removal rates of 0.5 g N?L^?1?day^?1 can be maintained when decreasing the temperature from 29 to 15 °C and when low nitrogen concentration and moderate COD levels are treated. Nitrite accumulation together with elevated NO and N₂O emissions (5 % of N load) were needed to favor anammox compared with nitratation at low free ammonia (<0.25 mg N?L^?1), low free nitrous acid (<0.9 μg N?L^?1), and higher DO levels (3–4 mg O₂?L^?1). Although the total nitrogen removal rates showed potential, the accumulation of nitrite and nitrate resulted in lower nitrogen removal efficiencies (around 40 %), which should be improved in the future. Moreover, a balance should be found in the future between the increased NO and N₂O emissions and a decreased energy consumption to justify OLAND mainstream treatment.     

Recovery of plant growth-promoting rhizobacteria from sodium alginate beads after 3 years following storage at 4 °C

Two plant growth-promoting bacteria, Bacillus subtilis and Pseudomonas corrugata, immobilized in a sodium alginate based formulation were evaluated for their survival, viability and plant growth-promoting ability after 3 years of storage at 4 °C. Populations of both of the bacterial isolates recovered from the immobilized sodium alginate beads were in the order of 10⁸ cfu g⁻¹. The plant-based bioassay indicated that the plant growth promotion ability of both of the bacterial isolates was equal to those of fresh broth-based formulations. The bacterial isolates retained the root colonization, and antifungal and enzyme activities in the alginate-based formulation during storage.     

Observation of two quenchers of chlorophyll fluorescence in chloroplasts at −196°C

Fluorescence induction at ?196°C has been monitored in chloroplasts rapidly frozen after poising at different redox potentials at room temperature. It was found that, as at room temperature, the initial level of fluorescence observed upon shutter opening (F_o), relative to the final level observed after 10 seconds of illumination (F_m) increased as the redox potential of the chloroplasts was lowered. Redox titration revealed the presence of two quenching components with E_m,7.8 at ?70 mV and ?275 mV accounting for approx. 75% and 25% of the variable fluorescence (F_v). Parallel observation of fluorescence yield at room temperature similarly gave two components, with E_m,7.8 at ?95 mV and ?290 mV, also accounting for approx. 75% and 25%. Simultaneous measurement of fluorescence emission at ?196°C at 695 nm and 735 nm indicated that both emissions are quenched by the same redox components.