Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria are aerobic microorganisms that can remove nitrogen under high-salt conditions, but their performance in practical applications are not satisfactory. As a compatible solute, trehalose helps microorganisms to cope with high salt stress by participating in the regulation of cellular osmotic pressure, and plays an important role in promoting the nitrogen removal efficiency of microbial populations in the high-salt environment. We investigated the mechanism of exogenous-trehalose-enhanced metabolism of HN-AD community under high-salt stress by starting up a membrane aerobic biofilm reactor (MABR) to enrich HN-AD bacteria, and designed a C₁₅₀ experimental group with 150 μmol/L trehalose addition and a C₀ control group without trehalose. The reactor performance and the community structure showed that NH₄⁺-N, total nitrogen (TN) and chemical oxygen demand (COD) removal efficiency were increased by 29.7%, 28.0% and 29.1%, respectively. The total relative abundance of salt-tolerant HN-AD bacteria (with Acinetobacter and Pseudofulvimonas as the dominant genus) in the C₁₅₀ group reached 66.8%, an 18.2% increase compared with that of the C₀ group. This demonstrated that trehalose addition promoted the enrichment of salt-tolerant HN-AD bacteria in the high-salt environment to enhance the nitrogen removal performance of the system. In-depth metabolomics analysis showed that the exogenous trehalose was utilized by microorganisms to improve proline synthesis to increase resistance to high-salt stress. By regulating the activity of cell proliferation signaling pathways (cGMP-PKG, PI3K-Akt), phospholipid metabolism pathway and aminoacyl-tRNA synthesis pathway, the abundances of phosphoethanolamine, which was one of the glycerophospholipid metabolites, and purine and pyrimidine were up-regulated to stimulate bacterial aggregation and cell proliferation to promote the growth of HN-AD bacteria in the high-salt environment. Meanwhile, the addition of trehalose accelerated the tricarboxylic acid (TCA) cycle, which might provide more electron donors and energy to the carbon and nitrogen metabolisms of HN-AD bacteria and promote the nitrogen removal performance of the system. These results may facilitate using HN-AD bacteria in the treatment of high-salt and high-nitrogen wastewater.
Nitrification ; Denitrification ; Trehalose ; Phosphatidylinositol 3-Kinases/metabolism* ; Heterotrophic Processes ; Salt Stress ; Nitrogen/metabolism* ; Aerobiosis ; Bioreactors/microbiology*

Trehalase is widely used in industrial fermentation, food, medicine and other fields. There is a lack of industrial varieties of trehalase with excellent performance in China. Moreover, the applied research on trehalase was not well conducted. In this study, a strain of Pectobacterium cypripedii was screened from nature, and the gene PCTre encoding an acidic trehalase was cloned and expressed in E. coli BL21(DE3). The highest enzyme activity reached 4130 U/mL after fermenting in a 5 L fermenter for 28 h. The enzymatic properties study showed that PCTre hydrolyzed trehalose specifically. The optimum pH and temperature were 5.5 and 35 ℃, respectively. 80% of the enzyme activity was retained after being treated at pH 4.0, 4.5, and 5.0 for 8 h, showing good acid tolerance. Moreover, it has good tolerance to organic solvents, 60% enzyme activity was retained after being treated with 20% (V/V) ethanol solution for 24 h. Furthermore, trehalose could be completely hydrolyzed within 16 h in a simulated fermentation system containing 20% (V/V) ethanol and 7.5% trehalose, with 500 U/L PCTre added. This indicated a good application potential for industrial ethanol fermentation.
Trehalase/metabolism* ; Trehalose/metabolism* ; Escherichia coli/metabolism* ; Ethanol/metabolism* ; Cloning, Molecular

This research aimed to study the effect of distillage recycling on ethanol fermentation, the key glycolytic enzymes and cell composition of the self-flocculating yeast. With the self-flocculating yeast SPSC01 and medium composed of 220 g/L glucose, 8 g/L yeast extract and 6 g/L peptone, continuous ethanol fermentation was carried out at the dilution rate of 0.04 h(-1) with a 1.5 L tank bioreactor. Fermentation broth was collected every 3 days, and ethanol and other volatile byproducts were removed by distillation, but the stillage with high boiling byproducts was recycled to prepare the medium instead of fresh water. The system was run for 20 days, during which ethanol and biomass concentrations in the effluent decreased continuously, indicating the significant inhibition of the high boiling byproducts accumulated within the system. Thus, the activities of the key enzymes of the glycolytic pathway: hexokinase, 6-phosphofructose kinase, and pyruvate kinase were analyzed, and it was observed that all of them were inhibited. Furthermore, the biosynthesis of the stress response metabolites glycerol and trehalose was investigated, and it was found that glycerol production that can protect yeast cells against osmotic pressure stress was enhanced, but trehalose biosynthesis that can protect yeast cells against ethanol inhibition was not improved, correspondingly. And in the meantime, the biosynthesis of the major intracellular components proteins and hydrocarbons was adjusted, correspondingly.
Bioreactors ; microbiology ; Ethanol ; metabolism ; Fermentation ; Flocculation ; Glycerol ; metabolism ; Glycolysis ; Hexokinase ; metabolism ; Industrial Microbiology ; methods ; Phosphofructokinase-1 ; metabolism ; Saccharomyces cerevisiae ; enzymology ; genetics ; metabolism ; Schizosaccharomyces ; enzymology ; genetics ; metabolism ; Trehalose ; metabolism ; Triticum ; metabolism ; Zea mays ; metabolism

By constructing the genomic DNA library of Meiothermus ruber CBS-01, the genes of trehalose phosphate synthase (TPS) and trehalose phosphate phosphatase (TPP) involved in trehalose synthesis were cloned. The genes were cloned into the plasmid pET21a, and expressed in Escherichia coli Rosetta gami (DE3). The activities of these two purified enzymes were confirmed by thin layer chromatography (TLC). Meanwhile, we tested the cellular compatible solutes of M. ruber CBS-01 under different environmental pressure, and found that under hyperosmotic pressure, this strain can accumulate trhalose-6-phosphate, but not trehalose. These results can give more insight to future research in the roles of TPS/TPP and TreS pathway.
Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; genetics ; metabolism ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Thermus ; enzymology ; genetics ; Trehalose ; biosynthesis

Ethanol tolerance of self-flocculating yeast SPSC01 was studied in a 3-L bioreactor under fed-batch culture. Yeast floc populations with the average sizes around 100, 200, 300, and 400 microm were obtained by adjusting the mechanical stirring rates of the fermentation system. When subjected to 20% (V/V) ethanol shock for 6 h at 30 degrees C, the remained cell viability was 3.5%, 26.7%, 48.8% and 37.6% for the aforementioned four floc populations, respectively. The highest ethanol yield 85.5% was achieved for the 300 microm flocs, 7.2% higher than that of the 100 microm flocs. The amounts of trehalose and ergosterol (including free ergosterol and total ergosterol) were positively correlated with the average size distributions from 100 to 300 microm. However, in the 400 microm flocs, the content of trehalose and ergosterol decreased, which coincided with its reduced ethanol tolerance compared to that of the 300 microm flocs. Furthermore, when subjected to 15% (V/V) ethanol shock at 30 degrees C, the equilibrium nucleotide concentration and plasma membrane permeability coefficient(P') of the 300 microm flocs accounted for only 43% and 52% respectively of those of the 100 microm and 200 microm populations. The effect of floc size distribution on the ethanol tolerance of the self-flocculating yeast strain SPSC01 was closely related to plasma membrane permeability. An optimal floc size distribution with the highest ethanol tolerance and ethanol production level could be obtained by controlling mechanical stirring speed of the bioreactor, which provides basis for the process optimization of fuel ethanol production using this self-flocculating strain.
Bioreactors ; microbiology ; Drug Tolerance ; Ergosterol ; biosynthesis ; Ethanol ; metabolism ; pharmacology ; Fermentation ; Flocculation ; Industrial Microbiology ; methods ; Particle Size ; Trehalose ; biosynthesis ; Yeasts ; drug effects ; growth & development ; metabolism

Though high concentration of glucose can benefit the survival of lyophilized human red blood cells, the high concentration of glucose can result in serious damage of red blood cells. This study was aimed to investigate the inhibitory effect of trehalose on damage of red blood cells induced by high concentration of glucose. After incubation with the high concentration of glucose buffer containing different concentrations of trehalose for three hours at 37 degrees C, the phosphatidylserine exposure and the osmotic fragility of cells were analyzed by flow cytometry and the lipid peroxidation of membrane was evaluated by TBA method. The results showed that the high concentration of glucose could lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage which were dependent on the glucose concentrations and incubation temperature. However, trehalose could effectively prevent the phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage induced by high concentration glucose. With increase of the trehalose concentrations. As the trehalose concentration increases, the phosphatidylserine exposure, maloni-aldehyde concentration and cell debris rate decreased gradually. In conclusion, the high concentration of glucose can lead to phosphatidylserine exposure, osmotic fragility increase, and lipid peroxidation damage of red blood cells. However, trehalose can inhibit the damaging effects of high concentration of glucose on red blood cells, which may be useful for the application of sugars to lyophilization of red blood cells.
Blood Preservation ; methods ; Erythrocyte Membrane ; drug effects ; metabolism ; Erythrocytes ; drug effects ; Flow Cytometry ; Glucose ; adverse effects ; Humans ; Lipid Peroxidation ; drug effects ; Membrane Lipids ; metabolism ; Osmotic Fragility ; drug effects ; Phosphatidylserines ; pharmacology ; Trehalose ; pharmacology

This study was aimed to evaluate the effect of benzyl alcohol on trehalose-loading red blood cells (RBCs). The RBCs were incubated in 10, 30, 50 and 100 mmol/L concentrations of benzyl alcohol-trehaloe solution at 4 degrees C for 24 hours. The hemolysis rate of loaded RBCs was detected by using cyanohemoglobin kit, the intracellular trehalose level were assayed by sulfate anthrone method. The results showed that the intracellular trehalose concentration in group with 100 mmol/L benzyl alcohol was 72 +/- 12.98 mmol/L, compared with that in groups of 10, 30 and 50 mmol/L, the statistical difference were significant (p = 0.000); the hemolysis rate of loaded RBCs in group with 100 mmol/L of benzyl alcohol was 17.99 +/- 3.75%, as compared with groups of 10, 30 and 50 mmol/L, the statistical difference was significant (p = 0.000). It is concluded that benzyl alcohol can enhance the intracellular trehalose concentration. As concentration of benzyl alcohol ascends, the intracellular trehalose concentration increases. 100 mmol/L benzyl alcohol may be proper for loading RBCs.
Benzyl Alcohol ; pharmacology ; Blood Preservation ; methods ; Erythrocyte Membrane ; metabolism ; Erythrocytes ; drug effects ; metabolism ; Freeze Drying ; methods ; Humans ; Trehalose ; metabolism ; pharmacology

This study was aimed to investigate the effects of DMSO on platelets during pre-treatment for lyophilization, including centrifugation, washing and loading trehalose. After pre-treatment for lyophilization, the expression of platelet membrane surface glycoprotein (GP) including CD62p and PAC-1 was analyzed by FCM before and after induction with thrombin, the mean platelet volume (MPV) and platelet maximal aggregation with several platelet inducers were investigated. The results showed that the expression rates of CD62p and PAC-1, as the platelet activation signs, increased and were 30.37% and 15.01% respectively in group without DMSO after pre-treatment. And their differences in comparison with control were statistically significant, but that of CD62p was 10.72% and PAC was 10.11% in group with DMSO, in comparison with group without DMSO respectively, their differences were statistically significant after diluting with DMSO, CD62p was re-expressed to 54.39% in group with DMSO and more than that in group without DMSO and lower than control statistically significant. PAC-1 was re-expressed to 49.28% in group with DMSO and more than that in group without DMSO (p<0.01) and reached to control. Platelet maximal aggregations induced by thrombin, restocetin and propyl gallate were 92.76%, 91.24% and 89.66 respectively in group with DMSO. These were closed to that in control group and in group without DMSO. But the aggregation induced by ADP was 34.33%, it was less than control (p<0.01) and more than that in group without DMSO (p<0.01). It is concluded that DMSO can inhibit the expression of CD62p and PAC-1 on platelet in vitro. But when diluted with plasma, platelets can express CD62p and PAC-1 induced by thrombin and be led to aggregate by several inducers, so the inhibitory effects of DMSO on platelet activation are reversible. DMSO play roles in inhibitor damage from platelet activation and cryoprotectant. This property of DMSO is very important in research of platelets lyophilization.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Cell Survival ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Freeze Drying ; Humans ; Platelet Activation ; drug effects ; physiology ; Trehalose ; blood ; pharmacology

The aim of this study was to search a procedure of platelet lyophilization and find a way of long-term storage of human platelets at normal temperature with smaller size and lighter weight, to be convenient to transport at long distance thus to meet the demands in accidents and war time. Human platelets were pretreated by freezing, the first and the second desiccation, and were added with reversible activation-inhibitors of platelets, DMSO and trehalose, then were rehydrated. At the same time, the recovery rate of platelets, platelet maximal aggregation induced by thrombin, coagulation of platelets, CD62p expression and PAC-1 expression were assayed. The results indicated that the recovery rate of the platelets was 56.29%. The platelet maximal aggregation induced by thrombin had no significant difference between lyophilized platelets and the fresh platelet-rich plasma (FPRP), but the aggregation of platelets induced by ADP or propyl gallate was decreased by 49.34% and 26.25%. Coagulation of the lyophilized platelets was not significantly different from FPRP. CD62p expression of the lyophilized platelets (42.36%) was higher than that in FPRP while PAC-1 expression was 2.12%. CD62p re-expression rate induced by thrombin was 50.88% and PAC-1 re-expression was 54.55%. It is concluded that the ability of recovered lyophilized platelets added with reversible activation-inhibitors, DMSO and trehalose to aggregate and coagulate has showed no significant difference as compared with FPRP. The reversible activation-inhibitors can decrease CD62p expression of lyophilized platelets, and may enhance their survival ability and prolongate survival time. Therefore the efficiency of lyophilizing platelets can be improved based on this freeze-drying procedure.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Cell Survival ; Freeze Drying ; methods ; Humans ; Trehalose ; pharmacology

The key points for better protection of trehalose in freeze-drying red blood cells (RBCs) are to resolve non-osmosis of trehalose to red blood cells and to make cytoplasmic trehalose to reach effective concentration. This study was aimed to investigate the regularity of loading RBCs with trehalose, screen out optimal loading condition and evaluate the effect of trehalose on physico-chemical parameters of RBCs during the period of loading. The cytoplasmic trehalose concentration in red blood cells, free hemoglobin and ATP level were determined at different incubation temperatures (4, 22 and 37 degrees C), different trehaolse concentrations (0, 200, 400, 600, 800 and 1000 mmol/L) and different incubation times (2, 4, 6, 8 and 10 hours), the cytoplasmic trehalose, free hemoglobin (FHb), hemoglobin (Hb) and mean corpuscular volume (MCV) in fresh RBCs and RBCs stored for 72 hours at 4 degrees C were compared, when loading condition was ensured. The results showed that with increase of incubation temperature, time and extracellular trehalose concentration, the loading of trehalose in RBCs also increased. Under the optimal loading condition, cytoplasmic trehalose concentration and free hemoglobin level of fresh RBCs and RBCs stored for 72 hours at 4 degrees C were 65.505 +/- 6.314 mmol/L, 66.2 +/- 5.002 mmol/L and 6.567 +/- 2.568 g/L, 16.168 +/- 3.922 g/L respectively. It is concluded that the most optimal condition of loading trehalose is that fresh RBCs incubate in 800 mmol/L trehalose solution for 8 hours at 37 degrees C. This condition can result in a efficient cytoplasmic trehalose concentration. The study provides an important basis for long-term preservation of RBCs.
Biological Transport, Active ; drug effects ; Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; metabolism ; pharmacology ; Erythrocyte Membrane ; metabolism ; Erythrocytes ; Freeze Drying ; Humans ; Osmotic Fragility ; Temperature ; Time Factors ; Trehalose ; metabolism ; pharmacology