Enterobacter cloacae and Bacillus drentensis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and 16S rRNA gene sequencing techniques, respectively. Subsequently, in a 2-year mung bean field trial, using a randomized complete block design with a split-split plot arrangement, we evaluated the two PGPR strains and two Si levels (1 and 2 kg ha−1), in comparison with control treatments, under three different saline irrigation conditions . The results indicated that salt stress substantially reduced stomatal conductance, transpiration rate, relative water content (RWC), total chlorophyll content, chlorophyll a, chlorophyll b, carotenoid content, plant height, leaf area, dry biomass, seed yield, and salt tolerance index. The PGPR strains and Si levels independently improved all the aforementioned parameters. Furthermore, the combined application of the B. drentensis strain with 2 kg Si ha−1 resulted in the greatest enhancement of mung bean physiology, growth, and yield. Overall, the results of this study provide important information for the benefit of the agricultural industry.The present study explored the eco-friendly approach of utilizing plant-growth-promoting rhizobacteria (PGPR) inoculation and foliar application of silicon (Si) to improve the physiology, growth, and yield of mung bean under saline conditions. We isolated 18 promising PGPR from natural saline soil in Saudi Arabia, and screened them for plant-growth-promoting activities. Two effective strains were selected from the screening trial, and were identified as  Salinity in agriculture is a global problem  and chloride (Cl−) ions in saline conditions  on crop growth and yield for plants under biotic and abiotic stress, but variable results have been reported for the beneficial effects of Si accumulation in plant tissues among plant species . Aliquots (1 mL) were inoculated on plates containing LB agar medium. The plates were incubated for 48 h at 27°C, and bacterial colonies were differentiated by their morphology, pigmentation, and growth rate. Fast-growing colonies of bacteria were selected and purified by streaking on a fresh agar plate of LB media. Pure cultures of the strains were preserved in glycerol at -40°C for subsequent use. The rhizobacterial strains were coded (P1–P18).The PGPR were isolated from the rhizosphere of mung bean growing at the King Abdulaziz University's agricultural research station , as described by Piromyou et al. . A dilut8–109 colony forming units mL−1). Mung bean seeds were surface sterilized as described by Torres et al.  under axenic conditions. An inoculum for each strain was prepared in sterilized conical flasks using LB broth. Each flask, containing 60 mL of sterilized LB broth, was inoculated with a selected bacterial strain and incubated for 72 h at 28 ± 1°C under shaking (100 rpm) conditions. Prior to seed inoculation, an optical density of 0.5 at 535 nm was achieved by dilution  production, in the presence or absence of its precursor l-tryptophan, was determined for the selected rhizobacteria, using the protocol described by Sarwar et al. . Exopolyhttp://eztaxon-e.ezbiocloud.net/). The sequence was aligned using ClustalX, and a phylogenetic tree was constructed by a distance (neighbor-joining) method using Mega4 software , and matrix solution, in triplicate, as described by Angelakis et al. . The aut−1, was applied to the plants.A 2-year (2014–15 and 2015–16) field trial was conducted at agricultural research station, Hada Al-Sham , Jeddah, Saudi Arabia under natural saline conditions to evaluate the performance of the selected PGPR strains and Si applications. Prior to the start of the experiment, soil samples were taken from the experimental site and analyzed for their physical and chemical properties Table . The expEnterobacter cloacae and Bacillus drentensis strains were prepared in broth media, as described above. For the inoculation of mung bean seeds, a slurry was prepared consisting of sterilized peat, a broth culture of the respective strains, and sterilized sugar solution (10%) in the ratio 5:4:1 v/v  was applied to the foliage using split doses of potassium silicate at 10 and 30 d after germination. For the Si treatments of 1 and 2 kg ha−1, a 0.6 and 1.2 g Si L−1 solution, respectively, was applied per sub-sub plot with a hand-operated sprayer. For the control treatment, pure water was sprayed. All other agronomic practices were kept uniform across the treatments throughout the experiment.Fresh cultures of Stomatal conductance was measured at the abaxial surface of the fully expanded leaves using a steady state leaf porometer upgraded model SC-1 Decagon,  during tgs, stomatal conductance; and VPD, vapor pressure deficit calculated from air temperature and leaf temperature provided by the porometer, using an online vapor pressure calculator .where, Fresh, fully developed leaves from the top of randomly selected plants were used to measure the relative water content (RWC) and electrolyte leakage (EL). The leaves were collected, sealed in plastic bags, and transported to the laboratory. After measuring the fresh weights, leaves were immersed in distilled water for 24 h in a refrigerator. Leaves were then placed over tissue paper for blotting, and the fully turgid weight was measured. Finally, the leaf samples were oven dried at 72°C for 24 h, after which the dry weight was recorded, and RWC was determined using the equation previously described by Teulat et al. .For the measurement of EL, the leaves were cut into uniform discs with a paper puncher and transferred into test tubes containing 25 mL deionized water. The test tubes were vortexed for 10 s, and the initial electrical conductivity (EC0) of the solution was measured. The test tubes were then refrigerated at 4°C overnight and assayed for EC1. Finally, the test tubes were autoclaved at 121°C for 20 min to determine EC2, and EL was calculated using the formula described by Yang et al. .Fresh leaves were used for chlorophyll measurements, following the protocol described by Arnon . Briefly2 areas in each sub-sub plot. Leaf area was measured according to Masle and Passioura  were measured from three randomly selected 1 massioura . Brieflyassioura  as:where, DWI, dry weight of stressed plant; DWI, dry weight of Si treated inoculated plant; DWC, dry weight of unstressed, non-Si treated uninoculated plants.P ≤ 0.05; Steel et al., The data collected from screening and field trials were analyzed using Statistix 8.1 software. The means were compared using the LSD test (P ≤ 0.05) higher for the inoculated mung bean seedlings than for the uninoculated (control) seedlings (Table E. cloacae) and P16 (B. drentensis) showed the greatest potential for promoting plant growth of mung bean seedlings.For most of the isolates, shoot and root fresh weights were significantly . The other closely related strains were Bacillus vireti and Bacillus soli. In the phylogenetic tree, P16 joined the cluster comprising the Bacillus drentensis species with 99% bootstrap support . A higher stomatal conductance (0.394 mmol m−2 S−1) and transpiration rate (3.644 mmol m−2 S−1) were noted for the P16 treatment than for the P6 and control treatments. For these parameters, there were significant (P ≤ 0.01) interactions between salinity × silicon (S × Si) and salinity × PGPR strain (S × P), whereas the interaction of silicon × PGPR strain (Si × P) was not significant (P ≤ 0.05) for stomatal conductance, but significant (P ≤ 0.01) for the transpiration rate. The mean values of the S × Si interaction (Table 7) indicated that the highest values for stomatal conductance (0.561 mmol m−2 S−1) and transpiration rate (6.808 mmol m−2 S−1) for the 2 kg Si ha−1 treatment was for the 3.12 dS m−1 saline irrigation, followed by the 5.46 and 7.81 dS m−1 treatments. Similarly, for the S × P interaction (Table 7), the highest stomatal conductance (12.72%), and transpiration rate (29.23%) was noted for the P16 strain in the 3.12 dS m−1 treatment. The lowest values for stomatal conductance and transpiration rate were noted for the control treatment with the 7.81 dS m−1 saline irrigation. Under the same salinity level, the stomatal conductance and transpiration rate were 70.42 and 124.73% higher, respectively, in the P16 treatment than in the control treatment. The Si × P interaction (Table 8) indicated that, compared to the uninoculated, non-Si control treatment, the highest improvement in transpiration rate was for the P16 strain with 2 kg Si ha−1, followed by the P6 strain.Stomatal conductance and transpiration rates decreased with increasing salinity Table . On the P ≤ 0.01) in the RWC of mung bean were observed for saline irrigation, Si, and bacterial inoculation  interaction of S × Si for RWC. The highest RWC (86.23%) was noted for the 2 kg Si ha−1 treatment with the 3.12 dS m−1 saline irrigation; the lowest RWC (55.52%) was noted for the non-Si control treatment with the 7.81 dS m−1 saline irrigation (Table 7).Significant differences  and for the strains (P16 < P6). Table P ≤ 0.01) interactions of S × Si and S × P for EL. The S × Si interaction means (Table 7) indicated a significant reduction (24%) in EL for the 2 kg Si ha−1 treatment under severe salinity stress (7.81 dS m−1), from that of the non-Si treatment. The S × P interaction revealed the highest EL for the uninoculated control with a 7.81 dS m−1 salinity level, and the lowest EL for the P16 inoculation with a 3.12 dS m−1 salinity level. For all levels of saline irrigation, EL was significantly lower for the P16 and P6 treatments than for the uninoculated control treatment. In particular, the P16 strain repressed the negative effects of the salt stress by decreasing EL by 46.77 and 46.25% in the 5.46 and 7.81 dS m−1 saline treatments, respectively, from that of the control.For EL, the individual effect of all treatments was significant (P ≤ 0.01) affected by all the treatments (Table −1 and the P16 strain being the most effective (Table Table 7) revealed that, for the 3.12 dS m−1 salinity level, the 2 kg Si ha−1 treatment and the P16 strain had the highest values for Chl a (0.787 g kg−1) and total chlorophyll (1.123 g kg−1), respectively. The highest reduction in Chl a and total chlorophyll content occurred under severe saline stress (7.81 dS m−1). However, at this salinity level, 2 kg Si ha−1 and P16 inoculation mitigated the adverse salt stress effects by increasing the Chl a and total chlorophyll content by 37.26 and 25.15%, respectively, over their respective control treatments. Moreover, there were significant (P ≤ 0.05) Si × P interactions for Chl a and Chl b, with plants treated with P16 and 2 kg Si ha−1 maintaining an increase of 12.67 and 51.45%, respectively, compared to the non-Si uninoculated control (Table 8).Photosynthetic pigments  were significantly (ts Table . An incrve Table . The meaP ≤ 0.01) reduction in plant height was observed for increasing levels of salinity (3.12 to 7.81 dS m−1). In contrast, plant height significantly (P ≤ 0.01) increased with increasing doses of Si. Likewise, P6 and P16 inoculations significantly (P ≤ 0.01) increased plant height by 14.48 and 27.17%, respectively, over that of the uninoculated control. Significant (P ≤ 0.01) interactions were observed for S × P and Si × P, whereas the S × Si and S × Si × P interactions were not significant (P ≤ 0.05). The interaction of S × P revealed that, for all salinity treatments, plant height was significantly higher for P16 than for P6 and the uninoculated control (Table −1). A marked decline in plant height, 35.21 and 28.59 cm, was noted for uninoculated plants at the 5.46 and 7.81 dS m−1 salinity levels, respectively. Furthermore, the Si × P interaction revealed that the P16 strain with the 2 kg Si ha−1 resulted in the highest plant height, whereas the lowest plant height was noted for the uninoculated, non-Si plants  reduced the leaf area of mung bean (Table P ≤ 0.01) S × Si and S × P interactions for the leaf area of mung bean  was for the 2 kg Si ha−1 treatment with 3.12 dS m−1 of saline irrigation. The lowest leaf area (192.16 cm2 plant−1) was observed for the 7.81 dS m−1 saline irrigation in plots where no Si was applied. Similarly, the S × P interaction (Table −1), and a lower leaf area for uninoculated plants under high salinity conditions (7.81 dS m−1). For the different salinity treatments, 3.12, 5.46, and 7.81 dS m−1, leaf area increased by 85, 56, and 126% for the P16 strain and by 26, 23, and 96% for the P6, respectively, over that of the uninoculated control.Mean values for the S × Si interaction Table  suggesteP ≤ 0.01) interactions were observed for S × Si and S × P for dry biomass production. The S × Si interaction revealed that the highest dry biomass was for the 2 kg Si ha−1 treatment under the low saline irrigation conditions, whereas the lowest dry biomass was observed for the 7.81 dS m−1 plots without Si (Table −1) noted for P16 plants in the 3.12 dS m−1 treatment, and the lowest biomass (1.658 t ha−1) noted for the uninoculated plants in the 7.81 dS m−1 treatment  decreased the seed yield of mung bean, whereas increasing rates of Si and rhizobacterial inoculation (P16 > P6) increased seed yield (Table P ≤ 0.01) S × Si and S × P interactions for the seed yields of mung bean.Salinity significantly (lation P1 > P6 inc−1) was recorded for 2 kg Si ha−1 under 3.12 dS m−1 salinity, and the lowest yield (0.46 t ha−1) was recorded for the non-Si control under 7.81 dS m−1 salinity. The 2 kg Si ha−1 treatment significantly improved the seed yield by 119% over that of the non-Si control, under 7.81 dS m−1 salinity. For all levels of salinity stress, 1 kg Si ha−1 had no significant effect on seed yield. The S × P interaction (Table −1) noted for the 3.12 dS m−1 treatment. Saline stress at 7.81 dS m−1 significantly reduced seed yields, and the lowest value (0.552 t ha−1) was recorded for the uninoculated control.The S × Si interaction Table  revealedP ≤ 0.01) affected by salinity, Si, and rhizobacterial strains (Table P ≤ 0.01), and Si × P (P ≤ 0.05) interactions were significant, whereas the S × Si × P interaction was not significant (P ≤ 0.05). The S × Si interaction  noted for the non-Si plots with 7.81 dS m−1 saline irrigation. Table −1 of saline irrigation, respectively, over that of the uninoculated controls. The Si × P interaction  was observed for the uninoculated, non-Si plants.The STI was significantly (ns Table . Moreoveon Table  showed t STI 0.23 noted fo−1) and two PGPR strains  were evaluated for their effect on the growth and physiology of mung bean under saline irrigation conditions. Our data suggest that the combined use of Si and PGPR is an effective approach for maximizing mung bean productivity on marginal land with saline water. The combined effects of Si and PGPR help the mung bean plants acclimatize to saline conditions by modifying a variety of plant physiological processes, including stomatal conductance, transpiration rate, water relations, and synthesis of photosynthetic pigments, thereby improving growth  and seed yield.In this 2-year field study, foliar spray of Si  activities in different crop plants  and P6 (Enterobacter cloacae) were the most promising for growth promotion  of mung bean. Both of these strains have IAA production, phosphate solubilization, and exopolysaccharide activity, and these traits might be the cause of the noted plant growth promotion  of mung bean under saline conditions. Similar to our results, Si applications have been noted to increase photosynthetic pigments in important agricultural crops under saline conditions, including wheat  under salinity stress has been recognized as an adaptation strategy to reduce water loss  and two promising PGPRs (E. cloacae and B. drentensis) from the harsh natural saline conditions of Saudi Arabia were assessed for their ability to mitigate the adverse effects of salinity. The present study revealed that 2 kg Si ha−1 with the PGPR strain B. drentensis was the most effective combination for improving gaseous exchange, water relations, photosynthetic pigments, growth, and seed yield for mung bean under saline irrigation conditions. Moreover, the combined use of Si and PGPR showed an additive effect and represents an improved strategy for the amelioration of salinity stress in mung bean. Our research suggests that the combined use of Si and PGPR in agricultural is a sustainable strategy for the alleviation of salinity stress in mung bean. Further studies are required to explore the effect of Si on microbial gene expression and plant biochemistry, and to test whether the application of Si and PGPR might be a general strategy to improve stress tolerance of other crops.In conclusion, the present study comprehensively quantified the effect of salinity on the physiology, growth, and yield of mung bean. Moreover, foliar sprays of Si (1 and 2 kg haSM, ID, SA, and SA designed and executed the experiments. MM, SM, and SA carried out the lab work regarding chlorophyll measurement, relative water content, and electrolyte leakage. MY and ID were involved in rhizobacteria isolation, characterization and identification. SM and ID performed statistical analysis and wrote the manuscript. HH, SA, and ZA provided throughout technical support and helped in concluding the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AbstractSpinonychiurus sinensissp. n., which has seven chaetae in the distal row of the tibiotarsi. It is placed in the genus Spinonychiurus due to two important characters: the two subsegments on Abd. III sternum and the absence of d0 on the head. This is the first report of the genus Spinonychiurus in China. The diagnosis of Spinonychiurus is broadened and the key to the world species is provided.A new collembolan species is described,  PageBreakSpinonychiurus was established by Onychiurus edinensis Bagnall, 1935, and it was assigned to the tribe Onychiurini Börner, 1901 as having compound vesicles on the postantennal organ. Thalassaphorurini Pomorski, 1998 by the structure of the furcal area and the distinct S-chaeta on the antennae. So far, only four species of the genus Spinonychiurus are reported throughout the world: Spinonychiurus edinensis, Spinonychiurus epaphius Kaprus’ & Tsalan, 2009 from Ukraine, Spinonychiurus subedinensis  from Spain and Spinonychiurus vandeli  from France.The genus Spinonychiurus Weiner, 1996 but having 7 chaetae in the distal row of tibiotarsi .Labial types are identified after Abbreviations used in descriptions and figures: – antennal segmentsAnt.,  – postantennal organPAO,  – thoracic segmentsTh.,  – abdominal segmentsAbd.,  – pseudocelluspso,  – pseudoporepsp,  – parapseudocellipsx,  – unpaired parapseudocellipsxm,  – microsensillumms,  – chaeta of row pp-chaeta,  – S-chaetaS,  – posterior S-chaeta on Abd. VSp,  – sensory organ of antennal segment IIIAIIIO.The pseudocelli and pseudopores formulae are the number of pseudocelli or pseudopores per half-tergum or half-sternum. The S-chaetae formula is the number of S-chaetae per half-tergum or half-sternum from head to Abd. VI.Taxon classificationAnimaliaPoduromorphaOnychiuridaeGenusWeiner, 1996Onychiurus edinensis Bagnall, 1935: 117.PageBreakPostantennal organ oval, with numerous compound vesicles perpendicular to the long axis; clubs of AIIIO smooth, ribbed or granulated; Ant. IV with differentiated S-chaetae; posterior pso on head present; chaeta d0 on head absent; S-chaetae on the body well marked; Abd. V tergum with or without spines; Abd. VI with one or two axial chaetae; anal spines present or absent; distal whorl of tibiotarsal chaetae as 7 or 11; Abd. III sternum divided into two subsegment; furcal rudiment as a finely granulated area with 4 small dental chaetae in two rows posteriorly, one manubrial row of chaetae present posteriorly to dental chaetae.Taxon classificationAnimaliaPoduromorphaOnychiuridaehttp://zoobank.org/BC4B8586-C7AE-4DB2-A1FB-E02B17174A8A43.0376°N, 128.9965°E), 3.Oct.2011, litter and soil, Berlese extraction, leg. Tang Xuguang.Holotype: female; paratypes: 3 females and 1 male on slides - China, Jilin, Changbai Mountain Range  and 6 basolateral  chaetae , 16  and 15  chaetae each Fig. . Unguis Genital fig with 9–11 chaetae in females, 32 chaetae in male. Male ventral organ absent. Anal valves with numerous acuminate chaetae; each lateral valve with a0, 2a1, 2a2; upper valves with chaetae a0, 2b1, 2b2, c0, 2c1, 2c2. Anal spines set on distinct papillae, 0.8 times as long as inner edge of hind unguis.Spinonychiurus in China.Named for the first recordof the genus Found in the coniferous forest.Spinonychiurus in two important characters: two subsegments on Abd. III sternum and the absence of d0 on the head. However, it does not fit the current definition of the genus as proposed by Spinonychiurus and broaden its diagnosis accordingly. The main diagnostic characters of all known species of the genus are given in The new species is closest to the genus PageBreak


We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry.This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial  Milk is recognized by its nutritional value as it presents in its composition an elevated concentration of calcium and proteins of biological value. It also has bioactive peptides with a protective function in human health, such as antibacterial, antiviral, antifungal, antioxidant, antihypertensive, and antithrombotic action , 2.Dairy whey, such as cheese whey and whey permeate, is widely used in the elaboration of new products due to its lactose, proteins, vitamins, and dietary minerals compositions –5. In adβ-galactosidase enzyme levels in intestinal walls. This enzyme is present in mammals during the breast-feeding period; however, for most of them, the β-galactosidase activity decreases after the said period, which characterizes primary hypolactasia and brings about symptoms of lactose intolerance. This disorder affects about 70% of the world adult population [However, the lactose present in milk, in dairy products and in whey from dairy industries, limits the consumption of these products by individuals who are lactose intolerant. Intolerance to this sugar is a result of the reduction in the pulation . Accordiβ-galactosidase enzyme, generating glucose and galactose monosaccharides, emerges as a key biotechnological process with application in the dairy industry. β-Galactosidase was first used in the preliminary treatment of milk used in the production of candies and fermented dairy products. This application leads to an enhancement of the sensorial and technological properties of the end products, such as higher sweetness, higher solubility, and decrease in fermentation period [The food industry has been seeking to develop low-lactose or lactose-free products. Thus, sugar enzymatic hydrolysis from the n period , 12.Another application for the hydrolysis process aims at adding value to whey, whose improper disposal poses a serious environmental problem. The number of microorganisms capable of metabolizing lactose as a source of carbon is smaller than the number of microorganisms capable of metabolizing glucose and galactose . Therefoβ-Galactosidases have animal, vegetable, or microbial  origins, but the microbial enzymes show a higher productivity, resulting in cost reduction. The β-galactosidases used in industrial scale must come from Generally Recognized as Safe (GRAS) microorganisms. Enzymes obtained from fungi (Aspergillus oryzae and Aspergillus niger) and yeasts (Kluyveromyces lactis and Kluyveromyces fragilis) show great commercial potential. The choice of the β-galactosidase source usually depends on the hydrolysis reaction conditions. The ones from fungi present an optimum pH between 2.5 and 5.4, being usually used for acid whey hydrolysis. The ones obtained from yeasts present optimum pH between 6.0 and 7.0, and its use is more adequate for milk and sweet whey lactose hydrolysis. Other important features for the use of the enzyme in industrial processes include thermostability and the elevated enzymatic activity at low temperature, which enables its application in conditions that do not alter the sensorial and nutritional features of milk and its derivatives [ivatives , 16.β-galactosidases of microbial origins.In this context, the work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases enzymes used, Lactomax F30 and Lactomax Pure, obtained from the Aspergillus oryzae (A. oryzae) and Kluyveromyces lactis (K. lactis) strains, respectively, were provided by Prozyn .The low fat powdered milk and the powdered cheese whey used for this paper were ceded by Brasil Foods S.A. . The cheese whey powder was provided by Arla Foods . The commercial β-galactosidase activities of the commercial enzymes were assayed using o-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate. The initial rate of formation of free o-nitrophenol (ONP) was recorded spectrophotometrically at 415 nm. One unit of enzyme activity (U) is defined as the amount of enzyme that liberates 1.0 μmole of o-nitrophenol per minute under assay conditions.The  A. oryzae or K. lactis  β-galactosidase in orbital rotation shaker , at 150 rpm, with temperatures of 10 and 55°C for the A. oryzae enzyme and 10 and 37°C for the K. lactis  β-galactosidase. These enzymatic concentrations corresponded to 0.1, 0.2, and 0.3 g/L of A. oryzae  β-galactosidase and to 0.37, 0.74, and 1.11 g/L of K. lactis  β-galactosidase. After 0, 1, 2, 4, 8, and 12 h of hydrolysis reaction, we collected samples that were submitted to a 100°C for 10 min, so as to inactivate the enzyme, and afterwards we determined the glucose concentration. All the experiments were performed in triplicate.We added 100 mL of low fat milk, cheese whey, or whey permeate reconstructed at 5% (m/v) of lactose to 250 mL Erlenmeyer flasks. The processes of enzymatic lactose hydrolysis were performed using 3, 6, and 9 U/mL concentrations for theEh) according to the following:Cglu   is glucose concentration, MMlac is molar mass of lactose, Cilac is initial lactose concentration (50 g/L), and MMglu is molar mass of glucose.We calculated the milk and dairy whey lactose hydrolysis efficiency  and absorbance reading in spectrophotometer  at 505 nm. The glucose concentration was established based on a calibration curve. The analyses were performed in triplicate.F test. In the significant models, the averages were compared to the Tukey test, in a significance level of 95% (p < 0.05) using the BioEstat 5.0 software.The glucose concentration results represent the average from three independent experiments, performed in triplicate. The statistical evaluation of the conversion rate of lactose in glucose, present in milk, cheese whey, and permeate, in the different conditions of enzymatic hydrolysis, was performed through the Analysis of Variance (ANOVA), and the significance of the model was verified by the  A. oryzae (at 10 and 55°C) and K. lactis enzymes (at 10 and 37°C), with a 12 h reaction. The enzymatic concentrations used were 3, 6, and 9 U/mL  was defined as the amount of enzyme that liberates 1.0 μmole of o-nitrophenol per minute under assay conditions).Figures β-galactosidase, A. oryzae (55°C), and K. lactis (37°C) was also evaluated in the hydrolysis process.The lactose hydrolysis processes of milk, whey, and permeate were performed at low temperature (10°C) for both enzymes, with the aim of not altering the sensorial and nutritional characteristics of the milk and dairy whey. The enzymatic optimum temperature effect of each commercial β-galactosidase enzyme has been used at temperatures under optimum value of reaction, in order to avoid bacterial multiplication in the milk, as temperatures around 37°C correspond to the optimum condition for the development of various deteriorative and pathogenic microorganisms. The enzymes activity is influenced by multiple environmental factors, among which temperature is one of the most important parameters, and may affect the tridimensional structure or the protein conformation [According to Campos et al. , the β-gormation . A. oryzae  β-galactosidase than for K. lactis  β-galactosidase. Figures  K. lactis enzyme, the glucose concentrations generated from the lactose hydrolysis are similar under both temperatures (10 and 37°C), regardless of the raw material.Comparing the hydrolysis process at 10°C and at the optimum temperature of each enzyme, we verified that, under the experimental conditions applied, the temperature variation effect was more expressive for A. oryzae  β-galactosidase was used, regardless of the studied raw material. For the K. lactis enzyme, the lactose hydrolysis results only presented the same behavior of the fungal β-galactosidase when the whey permeate was used as a substrate. The results were similar for the milk and cheese whey hydrolysis, regardless of the tested K. lactis enzyme concentration.Regarding the enzyme concentration used in the lactose hydrolysis process, we verified that the variation for this parameter had higher influence when the Kluyveromyces  β-galactosidases at 2°C for 72 h. The authors observed that the fourfold increase in the enzyme concentration doubled the hydrolyzed lactose concentration in the milk after a 12 h reaction.Enzyme concentration directly influences lactose hydrolysis, as was already observed in other papers , 20. Hor K. lactis  β-galactosidase in concentrations around 3, 6, 9, and 12 U/mL at 37°C for 30 h. The authors verified that the increase of the enzymatic concentration led to a conversion increase in the lactose present in the milk.Akgül et al.  analyzed A. oryzae  β-galactosidase the concentrations of glucose gradually increased throughout the whole hydrolysis period, reaching, in 12 h at optimum temperature, values close to the maximum theoretical conversion of lactose in its respective sugars. As for the K. lactis  β-galactosidase, we verified a gradual increase in glucose concentration during the first two hours of reaction, and after that, it remained practically constant throughout the evaluated process, reaching values of about 20 g/L of glucose.In Figures  A. oryzae (at 10 and 55°C) and K. lactis enzymes (at 10 and 37°C) after 2 h of reaction. The enzymatic concentrations used were 3, 6, and 9 U/mL.Tables The hydrolysis efficiency was evaluated in 2 h reaction taking into account the possible industrial application of the enzymes in the referred process.p < 0.05) was observed between the efficiencies of the lactose hydrolysis in milk, whey, and permeate when using 6 and 9 U/mL concentrations, for both enzymes at optimum temperatures and at 10°C. However, for the 3 U/mL concentration, efficiency was significantly lower (p < 0.05) for most of the conditions evaluated.No significant difference , at the end of 12 h of reaction, regardless of the enzyme concentration used. For milk lactose, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions.The results obtained indicate that at temperature of 10°C the


This study was conducted to determine the prevalence of a low appendicular skeletal muscle index (ASMI) using three cut-off points (mean ASMI-2SD of a gender-specific young reference group (aged 18–39 years), mean ASMI-1SD of a gender-specific young reference group, and the lower 20 percentile value of a gender-specific older group (aged ≥ 65 years)) in Korean older people and the relationship between ASMI and subjective health-related quality of life.This study utilized data acquired during the Korean National Health and Nutrition Examination Survey (KNHANES) from 2008–2011. Dual-energy X-ray absorptiometry body compositional data was obtained from a subsample of 6538 subjects  aged 18–39 and 4413 subjects  aged 65 years and older. The three definitions of low appendicular skeletal muscle and the EQ-5D-3 L-Korean descriptive system were applied to Korean older people.2), ASMI 20 (6.48 kg/m2), and ASMI 1SD (6.95 kg/m2). In women, ASMI 2SD (4.38 kg/m2) was the lowest, followed by ASMI 1SD (4.96 kg/m2) and ASMI 20 (5.33 kg/m2). Proportions of older subjects with a low ASMI using the three cutoff points were 9.7% (ASMI 2SD) and 40.9% (ASMI 1SD) in men, and 0.7% (ASMI 2SD) and 7.4% (ASMI 1SD) in women. By multivariate ordinal logistic regression analysis, men with a low ASMI had significantly high odd ratios for the three domains of mobility (p < 0.001), self-care (p = 0.005), and usual activities (p = 0.004) among the five domains of the EQ-5D and EQ-5D index (p = 0.010).For the ASMI cutoff points used, in men, the three cutoff points were ASMI 2SD  be used for older Koreans.The ASMI 2SD cut-off points for older Koreans, 6.09 kg/m Life expectancies are increasing in most ethnic groups and countries, as are the survivals of older people with impaired physical functions and disabilities , 2. AccoSarcopenia is a term applied by Irwin Rosenberg in 1989 to explain age-associated loss of muscle mass and muscle function . Thereaf2 (ASM/m2) [The EWGSOP, International Working Group in Sarcopenia (IWGS) and AWGS guidelines define the sum of the muscle masses of the four limbs as appendicular skeletal mass (ASM) to calculate appendicular skeletal mass index (ASMI) as ASM/height(ASM/m2) , 4, 8–10(ASM/m2) . PreviouThis study was conducted to determine the prevalence of low ASMI in accordance with three cutoff points in aged Korean (>65 years), and to explore the relationship between low ASMI and subjective health-related quality of life.This study was based on data obtained from the 2008–2011 Korean National Health and Nutrition Examination Survey IV and V (KNHANES), a nationally-representative survey conducted by the Korean Ministry of Health and Welfare. KNHANES was a nationwide cross-sectional survey conducted in 1998–2012 by the Korean Center for Disease Control and Prevention (KCDC). KNHANES used stratified multi-stage clustered probability sampling to reflect the non-institutionalized Korean population. Participants completed a questionnaire that consisted of a health interview survey, a health behavior survey, a nutrition survey, and a health examination survey. A whole body dual energy X-ray absorptiometry (DXA) scan were performed on individuals ≥20 years old from July 2008 to June 2009 and on individuals of ≥10 years old from July 2009 to May 2011 (excluding pregnant women), individuals with a height of ≥196 cm or a weight of ≥136 kg were excluded in accord with the KNHANES survey protocol. In addition, test results were treated as missing value in participants with implanted radio-opaque material  that could affect DXA results [After excluding those without a whole body DXA scan , 6538 subjects  and 4413 subjects  were included in the final analysis. Written informed consent was given by all participants and the protocol for KNHANES IV and V were approved by the Institutional Review Board of the KCDC . Current study did not require additional Institutional Review Board approval because the KNHANES data set is publicly available.2), fat mass (g), lean mass ), and fat percent (%) for whole body and anatomical regions. From this data, ASM (kg) was calculated by summing the muscle masses of the four limbs, assuming that all non-fat and non-bone mass is skeletal muscle. ASMI was defined as ASM/height2[In the KNHANES study, whole body DXA examinations were conducted with a QDR4500A apparatus . All participants wore light clothing and removed all jewelry and other items that could interfere with DXA. The KNHANES data includes values for bone mineral content (g), bone mineral density (g/cm/height2.The following exclusion criteria were applied to the 6538 eligible subjects (aged 18 to 39 years), a prior diagnoses or treatment for diabetes, stroke, angina, myocardial infarction, thyroid disease, arthritis, asthma, chronic obstructive pulmonary disease, depression, chronic renal disease, chronic viral hepatitis, liver cirrhosis, or any malignancy. In addition, subjects hospitalized for any reason during the past year were also excluded (n = 1835). A total of 4703  were recruited for the young (18–39 years) reference group. Low ASMI groups were defined using the following three cutoff point points: ASMI 2SD - the cutoff value of the lowest 2.28% in gender-specific young reference groups, ASMI 1SD - the cutoff value of the lowest 15.87% in gender-specific young reference groups, and ASMI 20 - the cutoff value of the lowest 20% in gender-specific older study participants.Unlike previous studies, ASMI values of the young reference group in the current study were not normally distributed. Because young women's data showed high kurtosis, we used approximate values  in place of 1SD and 2SD values, respectively. These approximate values can be used in non-normally distributed data, whereas values of 1SD and 2SD can be used in normally distributed data .To measure health-related quality of life, we used the European Quality of Life-5 Dimensions (EQ-5D) instrument of the EuroQol group . Validit2), normal weight (BMI: 18.5-22.9 kg/m2), overweight (BMI: 23.0-27.4 kg/m2), and obese (BMI ≥27.5 kg/m2) [The covariates included in the analysis were age, education, family income, body mass index (BMI), smoking status, alcohol consumption, chronic illness, and number of chronic illness. BMI was used to assess obesity as determined by the World Health Organization Asia-Pacific criteria: underweight (BMI <18.5 kg/m5 kg/m2) . Smoking5 kg/m2) . Chronicp values of <0.05. SPSS ver. 21.0  was used for the statistical analysis.Statistical analyses were conducted using the complex sample procedure because the KNHANES data set was selected using a representative, stratified, and clustered sampling method, not a random sampling method. The data in Table 2 and in the young women group was 5.67 ± 0.78 kg/m2. If ASMI in the young reference group had a normal distribution, the values of mean-2SD and the lowest 2.28% would have been the same. However, young men group (Kolmogorov-Smirnov p = 0.020) and the young women group (Kolmogorov-Smirnov p < 0.001) did not show a normal distribution due to high kurtosis. In the young men group, the mean-2SD value was 5.99 kg/m2 and the lowest 2.28% value was 6.09 kg/m2, and in the young women group corresponding values were 4.12 kg/m2 and 4.38 kg/m2, respectively. The difference between the two values was higher in the young women group (0.26 kg/m2 vs. 0.10 kg/m2). The ASMI cutoff points in older men were ASMI 2SD (6.09 kg/m2), ASMI 20 (6.48 kg/m2), and ASMI 1SD (6.95 kg/m2), and corresponding values for older women were ASMI 2SD (4.38 kg/m2), ASMI 1SD (4.96 kg/m2), and ASMI 20 (5.33 kg/m2)  men and women, respectively, however, according to cutoff point of ASMI 1SD, 40.9% and 7.4% of the older men and women, respectively, had a low ASMI. In addition, only older men with a low ASMI were found to have a low health-related quality of life.2 and 5.89 ± 0.66 kg/m2 in older Korean men and women, respectively, which are a little lower than those of previous studies. The New Mexico Elder Health Survey for Hispanic and non-Hispanic Americans conducted from May 1993 to September 1995 reported a mean ASMI was 7.7 ± 0.7 kg/m2 in older men and 5.9 ± 0.7 kg/m2 in older women [2 in older men and of 6.1 ± 0.8 kg/m2 in older women [2 in older men and 6.32 ± 0.6 kg/m2 in older women [2 and 6.4 ± 0.6 kg/m2 in older men and women, respectively [The means and SDs of ASMI were 7.14 ± 0.81 kg/mer women . The Heaer women . A Japaner women . Finallyectively .2 and 5.67 ± 0.78 kg/m2 in men and women, which are markedly lower than the values obtained in the American study, which reported mean ASMIs for a young reference group (18–40 years) of 8.6 ± 1.1 kg/m2 and 7.3 ± 0.9 kg/m2 in men and women [2 and 5.9 ± 0.8 kg/m2 in men and women [On the other hand, mean ASMIs in the young reference group in the present study were 7.81 ± 0.91 kg/mnd women . The prend women .In The New Mexico Elder Health Survey for Hispanic and non-Hispanic Americans, the mean ASMIs of older men and women were approximately at the 87 and 80 percentiles, respectively, of the young reference group . In our The proportion of older people with an ASMI two standard deviations below the mean ASMI of the young reference group also displayed a marked difference in the present study as compared with previously published values. In our study, older people with below ASMI 2SD were 9.7% in older men and 0.7% in older women, as compared to 13% at 61 to 70 years of age, 24% at 71 to 80 years of age, and 50% at aged 81 aged and older [2 in men and 5.21 kg/m2 in women and 7.09 kg/m2 in men and 5.27 kg/m2 in women, while a study in Taiwan used the lower 20 percentile values of gender specific healthy young age individuals [Interestingly, many studies on sarcopenia in Asian populations have not used the standard ASMI cutoff of two standard deviations below the mean ASMI of the young reference group. The ASMI 1SD cutoff points used in Japanese and Korean studies were 6.53 kg/mividuals , 23, 25.A number of reasons can be suggested for the low proportions of older people with an ASMI 2SD below the mean ASMI of young reference groups in the present study. First, the body types of Koreans are changing due to improvements in living standards. According to the 2010 Size-Korea survey of body measurement, the average height of Koreans increased steadily up to 2003 and remained constant thereafter, whereas the body types of Koreans have consistently changed. In that survey, the proportion of body shape of Korean 20s women changed to 7.3-head units tall as compared with 20 years earlier a 7.2-head units tall, and leg lengths of Korean women were nearly 2 centimeters longer than 30 years earlier . Second,2 to 4.38 kg/m2 in older women, and had we applied the real ASMI 2SD value (4.12 kg/m2) only four older women would have had a low ASMI.Because ASMI values of the young reference group in the current study did not show a normal distribution, we used alternative values that corresponded to lowest 2.28% and 15.87% values in the young reference group in place of 1SD and 2SD values, respectively. After making this adjustment, the cutoff point of ASMI 2SD increased from 4.12 kg/mIt is suggested that the existing sarcopenia diagnostic criteria using ASMI of a young reference group of the same gender and race is inadequate for Koreans. Considering clinical outcomes later in life, re-establishment of optimal ASMI cut-off points is needed.We performed multivariate ordinal logistic regression analysis to examine the association between the low ASMI group based on the three established ASMI cutoff points mentioned and EQ-5D determined health-related quality of life. Low ASMI had significantly associated with poorer outcome of three domains of mobility, self-care, and usual activities among the five domains of the EQ-5D in older men. However, in older women with a low ASMI, these correlations were not significant for any of the three ASMI cutoff points. These findings are consistent with previous finding of a stronger correlation between sarcopenia and functional decline and disability in older men . Additio2, women 5.33 kg/m2) is appropriate for older people for the assessment of low ASM, until an optimal cutoff point is determined. Further studies on country/population specific ASMI cutoff point are needed in different communities.Although the association between the low ASMI group based on ASMI 20 and any domain of EQ-5D was not significant in older women, the ASMI 20 cutoff point was significantly associated with EQ-5D in older men. On the other hand, the ASMI 2SD and ASMI 1SD cutoff points may not be adequate to diagnose sarcopenia in older Koreans because the proportions of older people with sarcopenia are quite variable. Studies are needed on the optimal ASMI cutoff point with respect to muscle function and clinical outcome for Koreans. We suggest that an ASMI 20 cutoff point  and was nationally representative, the ASMI distribution based on the young reference group can be considered reliable. Second, we performed analyses in a gender-specific manner. We found that the association between height-adjusted ASM and EQ-5D differed in older men and women, which is consistent with the findings of the recent FNIH study, in which height-adjusted ASM was found to be more useful for men and weight-adjusted ASM for women with respect skeletal muscle functions [Finally, to our best knowledge, this study is the first report of a significant association between low ASMI and health-related quality of life. Our finding implies a possible relationship between sarcopenia and health-related quality of life, however, further studies are required to confirm the relation.On the other hand, the present study has following limitations that warrant consideration. First, the study lacked investigation of cause-effect relationships because of its cross-sectional nature. Future prospective cohort studies are needed for such investigations. Second, previous studies analyzed ADL and IADL as dependent variables, whereas we used EQ-5D as a dependent variable. Although EQ-5D contains items similar to ADL and IADL, such as, mobility, self-care, and usual activities, direct comparisons might be inappropriate. Third, we did not consider muscle strength and muscle function and we used only height-adjusted ASM. The recent guidelines  recommend to diagnose sarcopenia as reduced muscle mass related to muscle strength and function such as grip strength and gait speed, not only reduced muscle mass itself. However, our KNHANES data set did not contain information on muscle strength and muscle function, thus we have analysed only muscle mass in this study. Further studies are needed using our suggested ASM cutoff point and the FNIH guidelines, which represent the state of the art, recommend BMI-adjusted ASM. Finally, we did not include physical activity as a potential confounder because KNHANES IV and KNHANES V had different items of measurement. Further studies on the topic are needed.2 was used for men and 4.38 kg/m2 for women, which lowers its clinical usefulness because of the extremely low determined prevalence in women. Hence, we suggest that the cutoff point of the lowest 20% of Korean older people  be used for older Korean people.Among Korean older people, the prevalence of low ASM was 9.7% in men and only 0.7% in women when an ASMI 2SD cutoff point of 6.09 kg/m


EMB8. Media ingredients and culture conditions were optimized by “one-at-a-time approach.” Starch was found to be the best carbon source at 5% (w/v) concentration. Glucose acted as catabolic repressor for amylase production. Salt proved critical for amylase production and maximum production was attained at 5% (w/v) NaCl. Optimization of various culture parameters resulted in 48.0 IU/mL amylase production, a 12-fold increase over that of unoptimized condition (4.0 IU/mL). α-Amylase was immobilized on 3-aminopropyl functionalized silica nanoparticles using glutaraldehyde as cross-linking agent. Optimization of various parameters resulted in 96% immobilization efficiency. Starch hydrolyzing efficiency of immobilized enzyme was comparatively better. Immobilized α-amylase retained 75% of its activity after 5th cycle of repeated use.Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Present work encompasses production optimization and nanoimmobilization of an  Demand for α-amylase is projected to further increase in coming years due to its use in diverse industrial sectors [α-Amylases from wide range of sources with distinct characteristics are available. Yet search continues for novel α-amylase to increase the realm of processes where it can be used. In this context, isolation and screening of extremophilic organisms for α-amylase of desired trait is a contemporary research area. Halophiles, a class of extremophiles growing under saline conditions, offer source of enzymes which not only are salt stable but also can function under conditions of alkaline pH, high temperature, and low water activity [α-amylase in bioprocesses presents the advantage to obtain optimal activities at high salt concentrations. Halophilic α-amylases also might be particularly resistant to organic solvents because they work under condition where water activity is low. sectors –5. α-Amyactivity –8. The u Natronococcus amylolyticus [ Haloferax mediterranei [ Haloarcula hispanica [ Halomonas meridiana [ Chromohalobacter sp. TVSP 101 [ Nesterenkonia sp. strain F [ Halomonas meridiana [ Halobacillus sp. strain MA-2 [ Bacillus sp. strain TSCVKK [Halophilic amylases have been reported from extreme halophiles such asolyticus ; Haloferterranei ; Haloarcispanica . Halomoneridiana ; ChromohTVSP 101 ; Nesterestrain F  are modeeridiana , Halobacain MA-2 , and Bacn TSCVKK  for enhaα-amylase. The immobilization of enzymes on solid support offers several advantages over the free enzyme, including easy recovery from the reaction medium, reusability, possibility of operation in continuous reactors, enhanced stability, and catalytic efficiency [α-amylase. Some examples involve reactive polymer films [Another aspect covered in this study is immobilization of ficiency . There her films , magnetier films , mesoporer films , and adser films . In receer films –25. Theyer films . Marinobacter sp. EMB8 was isolated during screening of Indian saline habitats [α-amylase was purified and found to be salt and solvent stable. It was used for synthesis of industrially useful maltooligosaccharides [α-amylase from Marinobacter sp. EMB8 were attempted. Bacterial growth and enzyme production are greatly influenced by the nutritional factors  and physical factors . A systematic investigation for effect of these factors on α-amylase production by Marinobacter sp. EMB8 has been undertaken in the present study. We herein also attempted to immobilize the halophilic Marinobacter sp. α-amylase on silica nanoparticles to obtain an active, stable, reusable preparation for effective starch hydrolysis.Amylase producer halophilic bacteriahabitats . The α-acharides . With th3-Aminopropyl functionalized silica nanoparticles and betaine were product of Sigma Chemical Company . Soluble starch and glutaraldehyde were obtained from Merck Specialties Pvt. Ltd. . Bovine serum albumin (BSA) was purchased from Sisco Research Laboratories Pvt. Ltd. . The media components were purchased from Hi-Media Laboratories . All other chemicals used were of analytical grade.Marinobacter sp. EMB8 used in present study was isolated from the Indian sea coast of Kozhikode  during screening of halophilic bacteria for industrially important hydrolases. Amylolytic activity of the culture was screened on starch nutrient agar plates [r plates . The isoμL of soluble starch  in sodium phosphate buffer  containing 1% (w/v) NaCl and 500 μL enzyme. Reaction was carried out for 20 minutes at 45°C. The reducing sugar released by amylolytic activity was measured by the reduction of 3, 5-dinitrosalicylic acid (DNSA). One unit of amylase activity was defined as the amount of enzyme releasing 1 μmol of maltose equivalent per minute from soluble starch under assay conditions. Protein concentration was estimated by dye binding method [Amylase was assayed following the method of Bernfeld  using stg method , using B Marinobacter sp. EMB8 from the slant, into 25 mL of medium (A) containing (g/L) starch, 10.0; peptone, 5.0; yeast extract, 5.0, with NaCl, 50.0; pH 7.0. Overnight grown culture (OD ~1.0) was used as inoculum. Media optimization for amylase production was carried out by “one-at-a-time approach” wherein single parameter was changed at a time while keeping others at a constant level. In order to see the effects of various nutritional and physical factors on amylase production, medium (A) was used as basal. Various carbon, nitrogen sources, and metal ions were varied, one at time, in the media. One percent of the mother culture was seeded as inoculum into 50 mL medium contained in 250 mL Erlenmeyer flask. The incubation was carried out at 35°C and 200 rpm for 72 h. The growth was monitored by recording the absorbance of the culture at 660 nm against uninoculated culture as blank.Mother culture was prepared by inoculating a loopful ofμm cellulose acetate membrane  was assayed. Effect of different parameters was monitored on the growth (A660 nm) and amylase activity.For checking the amylase activity, cells were harvested after 72 h by centrifugation at 10,000 ×g for 10 min at 4°C. The cell-free supernatant filtered through a 0.22 Different carbon sources , namely, glucose, fructose, maltose, lactose, sucrose, cellulose, dextrin, and starch, were supplemented in the medium containing (g/L) peptone, 5.0; yeast extract, 5.0; NaCl, 50.0. Medium was inoculated with 1% (v/v) mother culture and incubation was carried out at 35°C for 72 h at 200 rpm shaking.Effect of concentration of starch, the best utilized carbon source, was tested by incorporating it in different concentrations ranging from 0 to 10% (w/v). Other culture conditions were kept the same. Growth and amylase production were monitored as described above.2HPO4, 0.87; MgSO4·7H2O, 6.2; KCl, 0.75; NaCl, 50.0, with other nitrogen sources, namely, (NH4)2SO4, NH4Cl, urea, gelatin, casein enzyme hydrolysate, yeast extract, peptone, tryptone, combination of yeast extract and peptone, and corn steep liquor. All the nitrogen sources were incorporated at 1% (w/v) nitrogen content. Culture samples were processed for amylase assay and growth measurements similarly.The effect of various inorganic and complex nitrogen sources was studied by replacing yeast extract and peptone in the medium containing (g/L) starch, 50.0; KCasein enzyme hydrolysate was found to be utilized best among various nitrogen sources tested. In order to investigate the influence of casein enzyme hydrolysate concentration, its concentration was varied  in the medium.2HPO4, 0.87; MgSO4·7H2O, 6.2; KCl, 0.75, with pH 7.0 seeded with 1% (v/v) inoculum at 35°C and 200 rpm. For evaluation of effect of metals, a range of metal ions at concentration 0.1% (w/v) was added into medium containing (g/L) starch, 50.0; casein enzyme hydrolysate, 10.0; NaCl, 50.0, to see the outcome on growth and amylase production.The strain was grown in varying concentrations  of NaCl in the medium containing (g/L) starch, 50.0; casein enzyme hydrolysate, 10.0; K2HPO4, 0.87; MgSO4·7H2O, 6.2; KCl, 0.75; NaCl, 50.0 to different values, ranging from 6.0 to 10.0.The influence of pH on growth and amylase production was monitored by adjusting the pH of the optimized medium (designated as B) (g/L): starch, 50.0; casein enzyme hydrolysate, 10.0; KThe effect of temperature was studied by incubating culture flasks at various temperatures ranging from 25°C to 45°C. The medium B adjusted to pH 7.0 was used and other culture conditions were essentially the same.Varying inoculum sizes  of overnight grown culture were used. Other conditions were kept as optimized. The medium B shaking speed during incubation was varied from static to 250 rpm. Other conditions were kept as those optimized, namely, media B adjusted to pH 7.0, inoculum 1% (v/v), and incubation at 35°C for 72 h.2HPO4, 0.87; MgSO4·7H2O, 6.2; NaCl, 50.0, with pH 7.0 seeded with 1% (v/v) inoculum at 35°C and 200 rpm. Samples were withdrawn at different time intervals to measure the bacterial growth and amylase production as described above.Amylase production was carried out under finally optimized conditions, namely, medium containing (g/L) starch, 50.0; casein enzyme hydrolysate, 10.0; Kα-amylase activity was checked by adding 1% (w/v) of various anions  of sodium and potassium in the assay mixture.Effect of chloride ions on α-amylase on the functionalized silica  was performed in a sodium phosphate buffer  using glutaraldehyde as a cross-linking agent. For the immobilization, the NH2 groups of silica (15 mg/mL in buffer) were activated with glutaraldehyde . The mixture was incubated at 25°C for 2 h with continuous shaking at 200 rpm. It was further washed eight times with buffer to remove unbound glutaraldehyde. The activated nanosilica was incubated with 1 mL of α-amylase  for 2 h at 25°C under constant shaking at 200 rpm. The unbound enzyme was removed as supernatant by centrifuging at 8000 ×g for 10 min. The pellet which consisted of nanosilica containing immobilized α-amylase was washed by buffer until protein free supernatant was obtained. The immobilized α-amylase was finally suspended in sodium phosphate buffer  and stored at 4°C. Immobilization efficiency was calculated as follows:Covalent coupling of Conditions were optimized to attain maximum immobilization efficiency, by varying one-at-a-time: glutaraldehyde concentration ; nanoparticle concentration (7.5–45.0 mg); enzyme loading (45–180 IU); and effect of protectants maltose, starch, betaine, and BSA .α-amylase (2 IU/mL) in 150 mL flasks. The hydrolysis was carried out at 45°C with continuous shaking at 200 rpm for 8 h. The aliquots were taken out at various time intervals and the formation of the reducing sugar was quantitatively determined by the DNSA reagent.The starch solution was prepared as 2.0% (w/v) in sodium phosphate buffer  containing 1% (w/v) NaCl. For hydrolysis in the batch process, 20 mL of starch solution was mixed with soluble or immobilized α-amylase activity was determined each time. The activity recorded in the first cycle was taken as 100% activity, for calculating the residual activity after each successive usage.The starch hydrolyzing activity of the immobilized enzyme was evaluated by reusing the immobilized preparation after each use. After each cycle, the bound enzyme was separated by centrifugation, washed, and stored in the same buffer. It was then used for next starch hydrolysis cycle as described in previous section. The All the experiments were done in triplicate and the variation was within ±5%. Marinobacter sp. EMB8 used in present study was isolated by us from Kozhikode sea water, India. The isolate was Gram-negative rod and grew well at high salt concentrations  and pH range of 6.0–9.0. On the basis of salt requirement for growth, it could be placed under moderately halophilic bacterium according to Kushner's [ Marinobacter. The sequence has been submitted in GenBank, NCBI, USA, with accession number GU059908. Marinobacter sp. EMB8 culture has been deposited in Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India, with MTCC number 12013. The isolate secretes amylase as observed by zone of hydrolysis on starch agar plate. The amylase production was confirmed by growing the cells in starch containing medium. The conditions were optimized further for maximum production of amylase.Halophiles have been perceived as a potential source of industrially useful enzymes endowed with unique stabilities –33. Mariushner's  classifiDifferent carbon sources at 1% (w/v) concentrations were used to see their effect on growth and amylase production. Results are shown in  Halobacterium halobium [ Halomonas meridiana [ Micrococcus sp. [ Natronococcus sp. strain Ah-36 [ Halomonas meridiana [Starch has been observed as best inducer for amylase production in case ofhalobium  and Haloeridiana . Glucoseccus sp. , Natronoin Ah-36 , and Haleridiana .Concentration of starch was varied in medium to achieve optimum production. Amylase production increased as a function of starch concentration reaching maximum 15 IU/mL at 5% (w/v). No amylase production was detected in absence of starch. Starch at 5% (w/v) concentration was used as carbon source throughout further studies.Nitrogen sources were varied  to see the effect on growth and amylase production . ComplexSince the highest production was obtained with casein enzyme hydrolysate, the effect of its concentration on amylase production was further optimized. Amylase production was optimum in the medium with casein enzyme hydrolysate concentrations 1 and 1.5% (w/v). Casein enzyme hydrolysate concentration beyond this led to decrease in amylase production. Casein enzyme hydrolysate at 1% (w/v) concentration was considered best and used for further optimization. Halobacterium halobium [ Halorubrum xinjiangense [ Bacillus sp. strain TSCVKK [ Chromohalobacter sp. TVSP101 [Peptone in case ofhalobium  and Haloiangense ; combinan TSCVKK ; trypton TVSP101  are some Halomonas meridiana [ Bacillus sp. strain TSCVKK [ Chromohalobacter sp. TVSP101 [ Halobacterium halobium [Salt proved critical as amylase production was severely affected in absence of salt. Optimum production was observed in presence of 5% (w/v) NaCl . Amylaseeridiana ; 10% forn TSCVKK ; 20% for TVSP101 ; 25% forhalobium .2HPO4 was found to be best for amylase production. No growth and amylase production were observed in case of CuSO4, CoSO4, and HgCl2. Amylase production was increased by calcium chloride in Bacillus sp. strain TSCVKK [ Chromohalobacter sp. TVSP 101 [ Halobacterium halobium [ Halobacillus sp. strain MA-2 was best in presence of sodium arsenate, while copper sulphate decreased and lead nitrate did not affect the production [A range of metal ions at concentration 0.1% (w/v) were tested to see their effect on growth and amylase production. Magnesium sulphate in combination with Kn TSCVKK  and ChroTVSP 101 . In a dihalobium . Amylaseoduction .Amylase production was observed in the pH range of 6.0–9.0. Optimum production was observed at pH 7.0 and 7.5. Growth as well as enzyme production was observed at alkaline pH suggesting the haloalkaliphilic nature of bacteria. Halobacillus sp. produced maximum amylase at pH 7.8 and temperature of 30°C [ Bacillus sp. strain TSCVKK, amylase production was maximum at 30°C and pH 8.0 [ Chromohalobacter sp. TVSP101 optimum conditions were pH 9.0 and 37°C [Different temperatures in range of 25–45°C were tried to observe effect on growth and amylase production. Optimum enzyme production level was attained at 35°C. Among other halophiles, of 30°C . In cased pH 8.0 . For Chrand 37°C . In geneComparable amylase production was observed for inoculum size in range of 0.5 to 5% (v/v) with optimum being at 1% (v/v). Marinobacter sp. was grown under optimized conditions with varying shaking speeds. Amylase production increased with increasing shaking speed, optimum achieved at 200 rpm. Interestingly, growth and amylase production were also observed under static condition. Effect of shaking speed and inoculum size on amylase production has been less investigated in halophilic bacteria. Halobacillus sp. was found to produce maximum amylase at shaking speed of 200 rpm [Aeration is very critical for growth and metabolism of aerobic microbes. In order to check the effect of shaking speed on biomass and amylase production, the 200 rpm . Marinobacter sp. EMB8 was carried out under finally optimized culture conditions. Amylase production was growth-dependent reaching maximum in 54 h as illustrated in  Halomonas meridiana [ Bacillus sp. strain TSCVKK [ Nesterenkonia sp. strain F [Amylase production byeridiana , Bacillun TSCVKK , and Nesstrain F .Media optimization for efficient amylase production led to twelvefold increase in production over unoptimized conditions. Amylase production level was quite good as compared to previously reported levels among halophiles. Marinobacter sp. EMB8. Study also revealed influence of various culture conditions on halophilic amylase production. The starch concentration, nitrogen source, and metal ions were critical and caused significant increase in yield.Present study is important from the viewpoint of low amylase production level secreted in halophiles. Significant increase was attained forα-amylases [ Marinobacter sp. α-amylase activity was checked in presence of sodium and potassium salt of varying anionic groups. Their effect on activity is illustrated in Increasing evidence is gathering to support that anions especially chloride play a critical role in activating amylases . To valiα-amylase was found to be activated by chloride ions. Bromide and iodide ions which are of comparable size to chloride ions also acted as activators but to a lesser extent. Fluoride ions which are of smaller size were not able to do so. Other anions comparable to chloride ions in size such as acetate, nitrate, and azide also activated α-amylase to varying degrees. Chloride ions are reported to act as allosteric activator in case of human as well as halophilic α-amylases such as Pseudoalteromonas haloplanktis. Binding of chloride ions leads to interaction with catalytic residues and ultimately activation of α-amylase activity [The activity , 41.In order to explore viable application of this enzyme, it was thought to use in immobilized form. It was immobilized on silica nanoparticles. The choice of silica as a matrix for immobilization was dictated by the fact that they are chemically inert and biocompatible. Also, silica nanoparticles can be easily functionalized , 43. Theα-amylase is loosely bound through weak forces and washing leads to enzyme leaching in the washing fractions.The adsorption method was not very effective as only 12% immobilization efficiency was attained and most of the activity came out in washing as unbound enzyme. This was not surprising as α-amylase (termamylaze) on silica nanoparticles. In another study Bacillus amyloliquefaciens  α-amylase immobilized on polyaniline-assisted silver nanoparticles through glutaraldehyde coupling retained 83% of its activity [Covalent coupling placed the enzyme firmly on the silica nanoparticles and about 3.5-fold more immobilization efficiency was attained at 2.5% (v/v) glutaraldehyde concentration. The optimum immobilization efficiency was enhanced by 1.0- to 2.0-fold by glutaraldehyde functionalization in case of cholesterol oxidase immobilization on silica-coated magnetic nanoparticles . Glutaraactivity .α-amylase and subsequent activity loss during immobilization. Results in α-amylase and helped 11% and 24% increase in immobilization efficiency, as compared to that of control (in absence of any protectant).Immobilization was carried out in presence of substrate starch, product maltose, neutral protein feeder BSA, and halophilic protein protectant betaine to protect the structure of This effect can be attributed to the fact that starch as substrate may have bound to active site and protected the part from denaturation during glutaraldehyde coupling. Maltose is also a product, albeit less effective. Varying concentration of starch as protectant showed that 0.5% (w/v) was sufficient to give maximum protective effect.α-amylase was 90 IU. Finally optimized conditions are summarized in The amount of nanoparticles used for immobilization is critical because the immobilization efficiency will largely depend on the available surface area. In our case, 15 mg nanoparticle was found to be optimum. Finally the conditions of immobilization were optimized in terms of optimum enzyme loading. The optimum concentration of Km and Vmax of immobilized enzyme remained unchanged and matched with free α-amylase properties as investigated previously [The enzymatic properties, namely, temperature, salt, and pH optima, eviously .α-amylase, the starch hydrolysis was attempted and compared with free enzyme. Results are depicted in  Marinobacter sp. EMB8 α-amylase produces maltotriose and maltotetraose upon starch hydrolysis. Maltotriose and maltotetraose rich maltooligosaccharides are more desirable in bread making industries due to their better antistaling properties [Considering the application of immobilized operties .Reusability potential of immobilized preparation was also explored. The preparation could be used effectively for 2 cycles; thereafter each cycle led to partial loss of activity. It retained 48% of its activity after 8th cycle. Bacillus amyloliquefaciens  α-amylase immobilized on polyaniline-assisted silver nanoparticles hydrolyzed only 27% more starch as compared to free enzyme. The preparation could be used 10 times and 20% loss in activity was recorded [ Bacillus alcalophilus  α-amylase immobilized on iron-oxide magnetic nanoparticles.In our opinion, it may not be the loss of activity but it may be loss of nanoparticles suspension going into washing each time. Therecorded . Mukherjrecorded  reportedα-amylase was immobilized on functionalized silica nanoparticles. Under optimized conditions, 96% immobilization efficiency was obtained. Although the properties of immobilized enzyme remained the same as that of native, it gained in terms of reusability in repeated cycles. The preparation was used for hydrolysis of starch. It is worthwhile mentioning that highlight of the enzyme was to make maltotriose and maltotetraose rich oligosaccharides. Such maltooligosaccharides are highly desirable for application in baking industries due to better antistaling properties.Halophiles suffer from the drawback of low level of enzyme production, which often limits their applications as source for industrial enzymes. The optimization of culture conditions for achieving maximum amylase production was carried out. Optimization led to about 12-fold increase in the production. The


Trypanosoma congolense. PCR confirmed a Trypanosoma congolense forest-type infection. Treatment consisted of six injections of pentamidine at 48-hour intervals. Trypanosomes had disappeared from the blood smears four days following the first injection. Clinical improvement was correlated with the normalization of laboratory values. The infection relapsed twice and the dog was treated again; clinical signs and parasites disappeared and the dog was considered cured; however, 6 years after this incident, serological examination by ELISA T. congolense was positive. The status of this dog (infected or non-infected) remains unclear. Hypoglycaemia was the most notable clinical feature in this case. It was spectacular in its severity and in its refractory nature; glucose administration seemed only to feed the trypanosomes, indicating that treatment of hypoglycaemia may in fact have been detrimental.A 20 kg German shepherd dog was presented to a French veterinary teaching hospital for seizures and hyperthermia. The dog had returned 1 month previously from a six-month stay in Senegal and sub-Saharan Africa. Biochemistry and haematology showed severe hypoglycaemia (0.12 g/L), anaemia and thrombocytopenia. Despite administration of large amounts of glucose (30 mL of 30% glucose IV and 10 mL of 70% sucrose by gavage tube hourly), 26 consecutive blood glucose measurements were below 0.25 g/L (except one). Routine cytological examination of blood smears revealed numerous free extracytoplasmic protozoa consistent with  The dog had returned 1 month previously from a six-month stay in Senegal and sub-Saharan Africa. She had been properly vaccinated and dewormed. The rabies antibody titre was 7.92 IU/mL, above the value required for re-entry to Europe. The owners reported lethargy, reduced appetite, weight loss and fever peaks > 40 °C for 6 weeks. The dog had no history of epilepsy.1.2.Cordylobia anthropophaga), but no ulcers. A tick  was identified on the coat. Superficial lymph nodes were enlarged, especially the popliteal lymph nodes. There was no ocular anomaly.At presentation, the dog was cachectic, sleepy but responsive, sternally or laterally recumbent , with hy1.3.Seizures led to suspicion of a metabolic disorder, such as hypoglycaemia. Encephalitis was also suspected; rabies was considered. Epilepsy was excluded because of the associated symptoms. Poisoning was excluded because of the duration of symptoms. Fever and lymph node enlargement led to increased suspicion of systemic infectious diseases like leishmaniasis, ehrlichiosis, borreliosis, anaplasmosis or babesiosis. Due to the travel history, infection by an indigenous African infectious agent was considered. Lymphoma was not excluded.1.4.9/L, reference 200–500 × 109/L). Blood type was DEA 1.1+. Other abnormalities included hypoalbuminaemia , hyperglobulinaemia  and hypokalaemia . Lactate, blood gas, alkaline phosphatase, alanine aminotransferase, bilirubin, urea, creatinine, prothrombin time and activated partial thromboplastin time were within reference ranges. Urinalysis was unremarkable. Systolic, diastolic and mean arterial blood pressures were within normal values. A blood smear was prepared at the time of blood sample collection and stained with Wright-Giemsa (Diff Quick). The lymph nodes were aspirated and stained.Routine biochemistry and haematology showed severe hypoglycaemia , normocytic , normochromic , hyporegenerative , anaemia  and thrombocytopenia  confirmed a Trypanosoma congolense forest-type infection. All the other taxa-specific PCRs were negative .Routine cytological examination of the blood smears revealed numerous free extracytoplasmic protozoa . The parngolense . The blo1.6.Trypanosoma congolense does not infect humans and the vector, the tsetse fly, is not present in France. No preventive measures were taken for the medical staff, except, as for any patient, not to handle biological fluids without gloves.1.7.The dog was transferred to the intensive care unit (ICU). On admission to the ICU, two catheters were placed, one for fluid administration and drug injection, the other for blood collection. A naso-oesophageal tube was placed for sucrose administration.1.7.1.12/L; thrombocytes: 135 × 109/L), but the dog remained in lateral or sternal recumbency, unable to get up, sleepy, almost slumbering.Anaemia, thrombocytopenia, tachypnoea and tachycardia led to transfusion of 10 mL/kg fresh whole blood. Mucous membranes became more pink, heart rate and respiratory rates returned to normal values and biological parameters improved ; no discrepancy was found between them. The dog was placed on a 5% glucose solution with 20 mmol/L KCl at a rate of 1 mL/kg/h, except during transfusion. The severe hypoglycaemia was treated with a 30 mL of 30% glucose IV bolus injected slowly at the injection site of the maintenance solution to prevent phlebitis. Ten millilitres of sucrose 70% (7 g/10 mL), Canadou®, a sugar syrup used for sweetening cocktails, was administered by gavage tube.On day 1, at 1 PM, glycaemia was only 0.12 g/L and the dog had a new seizure. The first measurements and most of the very low measurements were carried out simultaneously with a glucometer and a calibrated laboratory analyser  and 60 mL of fluids/hour (3 mL/kg/h). The dose of glucose administered was approximately two times that usually recommended. Despite this support, no clinical or physiological improvement was seen. Despite the lack of effect on glycaemia, glucose supplementation was continued to prevent the irreversible or even fatal neurological effects associated with profound hypoglycaemia. At midnight, the dog presented with head-shaking, the glycaemia then being 0.22 g/L. At 4 PM, glycaemia was undetectable  with the glucometer (the detection limit was 0.1 g/L).At 4 PM, glycaemia was 0.63 g/L. At 5 PM, glycaemia was 0.2 g/L. From 6 PM until 3 PM on day 2, the dog had glycaemia measurements every hour, i.e. 26 measurements, all below 0.25 g/L, with the exception of one of 0.36 g/L at 11 PM . After e®, at a dose of 2.5 mg/kg, a glucose-elevating drug used to treat refractory hypoglycaemia induced by insulinoma. Glycaemia increased. Glycaemia was 0.2 g/L at 5 PM, 0.32 g/L at 6 PM, 0.53 g/L at 7 PM, 0.61 g/L at 8 PM, 0.57 g/L at 9 PM and 1.08 g/L at 10 PM. At that time, the dog was standing upright and was able to walk. Supplemental glucose was then limited to a maintenance solution of 5% glucose at 1 mL/kg/h supplemented with K+ and 10 mL of 70% sucrose by gavage tube. The next four readings were nearly normal: 0.87 g/L at 11 PM, 1.18 g/L at 0:00 on day 3, 0.87 g/L at 2 AM, but by 4 AM glycaemia had dropped to 0.28 g/L. The initial treatment every hour and diazoxide every 12 h were insufficient to restore normoglycaemia, with values remaining around 0.4 g/L all of the third day.At 6 PM on day 2, the dog received diazoxide, ProglycemEvery hour, after glycaemia and body temperature measurements, the dog’s position was shifted in order to prevent pressure ulcers. Arterial blood pressures, pulse and respiration rates taken every hour were always within normal limits.1.8.®. It was ordered, and arrived on day 3. Treatment consisted of six injections at 4 mg/kg at 48-hour intervals [Animal trypanocides are not available in metropolitan France; as a result, a human product was used. The only drug quickly available was pentamidine, Pentacarinatentervals . On day 1.9.12/L), hypoglycaemia (0.68 g/L) and thrombocytopenia (42 × 109/L). Many trypanosomes were present on the blood smears. Ultrasound revealed marked splenomegaly with multinodular parenchyma and a hypoechoic pattern, consistent with reactive splenitis. A second transfusion was performed. Thirteen glycaemia measurements during the first 24 h were all low, with a minimum of 0.31 g/L and a maximum of 0.59 g/L  and thrombocytopenia (120 × 109/L). Again, parasites were visible on the blood smears. A third transfusion was performed. Again, three intramuscular injections of 4 mg/kg pentamidine were given, but at 10-day intervals. The dog came back for monthly examinations for 3 months, upon which clinical examinations, biochemistry and haematology were within normal ranges.Three weeks after the first relapse, or 7 weeks after the first admission, the dog again presented, after a seizure. Haematology still showed anaemia  was positive. PCR was negative.Six years after this incident the dog was presented for a physical examination prior to another trip to Senegal; she was healthy, no parasites were found on a blood smear, but serological examination by ELISA 2.2.1.Trypanosoma species or types described in carnivores worldwide [T. cruzi, responsible for American trypanosomosis or Chagas disease, is not present in Africa. T. evansi is a trypanosome mainly found in camels in Senegal. Dogs are rarely found to be infected with T. vivax, although there have been some reports in Cameroon [T. congolense and two subspecies of T. brucei: T. brucei brucei and T. b. gambiense [T. brucei and T. evansi were ruled out based on morphologic criteria.There are six orldwide . T. cruzCameroon . The majambiense , 40. In T. congolense are described (from the most pathogenic to the least): T. congolense savannah-type is most often found in cattle, in which it produces a rapidly fatal or severe infection, whereas T. congolense forest-type causes more chronic infection in cattle, but also in dogs. T. congolense kilifi-type is rarely reported, since it usually causes an asymptomatic infection [Three types of nfection . These pnfection .Nannomonas Trypanosoma sp. was characterized by microscopic observation of the parasites; a definitive diagnosis of T. congolense forest-type was reached using PCR sequencing confirmation  reinforces the impression that symptoms were due mainly to hypoglycaemia.Hypoglycaemia was spectacular in its severity: most values were <0.4 g/L and often close to 0.2 g/L, which in general is not compatible with life. However, the clinical effects were less severe than the laboratory values would suggest. At 4 PM on day 1, blood glucose was undetectable, yet the dog was not comatose or convulsing.The most striking observation was its refractory nature. There was a total lack of response to glucose administration. Severe hyperglycaemias are sometimes reported, but intravenous or oral glucose administration always causes an increase in glycaemia, at least temporary. Every hour, this dog received 7 g of oral sucrose, 10 g of IV glucose and rich food, for a total of almost 20 g of sugar every hour (1 g/kg/h), or 500 g of pure sugar per day. In total, over the four days that hypoglycaemia lasted, the dog received 2 kg of pure sugar , without any effect. The same phenomenon happened again during the first relapse, when the dog received almost 1 kg of pure sugar in two days. To our knowledge, such severe hypoglycaemia in the context of such aggressive glucose supplementation has never been reported in the veterinary literature.Plasmodium falciparum, attributed in part to large glucose requirements of malaria parasites [The origin of this severe and refractory hypoglycaemia was a crucial diagnostic question. Sepsis-induced hypoglycaemia is usually less pronounced, and not refractory. The hypothesis of rebound hypoglycaemia was rejected, because we never observed hyperglycaemic peaks. We did not measure insulin levels. Severe hypoglycaemia and hyperinsulinaemia have been observed in human patients infected with arasites . In our We found old studies in the 1930s that seem to support this hypothesis. Trypanosomes use enormous amounts of sugar for their metabolism; they consume twice their own mass of sugar within 24 h . InsulinThese old experimental studies were reinforced by two clinical observations in human patients.In a case reported in the United States to CDC internal records, a diabetic patient with African trypanosomosis required less insulin during the acute phase of his illness .in vitro utilization of glucose by trypanosomes. They drew two blood samples simultaneously. The first sample was placed on ice and carried to the laboratory; the glucose level was 1.03 mg/L. In the second sample, not placed on ice and analysed at the same time, the glucose level was only 0.48 mg/L. At the same time of venepuncture, in a capillary blood specimen, the blood glucose level, given by a rapid assay method (Accucheck®), was 1.07 mg/L. In our case, most measurements were performed with a glucometer, immediately after the sample was drawn, or with an analyser located in the ICU, so the possibility of spurious hypoglycaemia is improbable. However, this case emphasizes the large consumption of glucose by trypanosomes, even ex vivo.Another case observed in the United States described severe African trypanosomosis with spurious hypoglycaemia in a 58-year-old woman returning from Kenya and Rwanda . Despite2.3.Trypanozoon) in camels; it is not known as an active trypanocide against the sub-genus Nanomonnas, so it would not be suitable for the present case. Nifurtimox, a human drug, may be found in hospitals but rather targets Trypanosoma cruzi, and was not fully validated against Trypanozoon parasites; moreover, it may not be well suited for animal treatments. Pentamidine was discovered accidentally in 1937 in the course of research on blood sugar lowering agents which have the ability to disrupt the parasite’s energy metabolism [T. brucei ssp. (Trypanozoon). It was not evaluated against T. congolense in animals, and as a result, there are no data available on its efficacy against T. congolense or on resistance. Diminazene aceturate is not available in France and is thought to be toxic in dogs. However, it is currently used in dogs in Africa and Asia respectively against T. congolense and T. evansi [®), the standard veterinary trypanocide, in the treatment of T. brucei brucei infection in dogs [®) requires only a single intramuscular injection at 1 mg/kg, but is only available in Africa. Isometamidium is used every 2 months for chemoprophylaxis in military dogs in sub-Saharan Africa [There are no trypanocides currently available in metropolitan France although some of them may be found. Melarsomine dihydrochloride, sold for the control of heart worm under the registered name Immiticide, is a trypanocide also sold in Africa under the registered name Cymelarsan for the control of surra  an extravascular refuge of the parasite , (ii) insufficient treatment  or (iii) parasite chemoresistance. In the last two cases, the relapse generally occurs within 2–4 weeks, due to the fact that some of the parasites have not been killed. In our study, the treatment procedure was carefully applied and is considered to be reliable in terms of trypanocide quality, quantity and administration route. In this case, after a first series of six pentamidine administrations, the very early relapse observed is suggestive of parasite chemoresistance to pentamidine. Indeed, the second series of treatments was also followed by a very early relapse, which confirms the fact that some of the circulating parasites were not killed by the trypanocide treatment. More problematic is the fact that the parasite disappeared after the third treatment at the same dose, but applied at 10-day intervals. It is hypothesized that repeated treatments, that proved to be sub-curative, may have enhanced the immune system and have finally led to either a self-cure or a clinical cure with sustainable control of the parasitaemia down to undetectable levels. Iterative treatments that proved to be sub-curative would then be a way to achieve a curative treatment protocol. However, such situations reveal pre-existing drug resistance and would even increase the selection of more resistant strains which would be another important concern in enzootic countries.T. congolense (and PCR). A successful curative treatment would most probably lead to negative seroconversion within 6 months post-treatment, as is usually observed with African trypanosomosis in other animals.The actual status of the dog in our case is questionable. There are no specific data available on the persistence of antibodies to trypanosomes in dogs. However, based on the observations made in livestock , persistT. congolense does not affect humans; and (ii) the parasite requires a vector not present in France. However, dogs can be infected with T. brucei that infect humans and mechanical infection can occur in areas where tsetse flies are not present. Quarantine after return from endemic countries is sometimes recommended [In the present case, the risk of transmission to humans was low, since (i) ommended . Quarantommended . Our dog2.4.Trypanosoma congolense varies greatly with type. Therefore, PCR analysis of whole blood was performed to correctly identify this Trypanosoma sp. and type. The sensitivity of PCR is two to three times higher than microscopic observation of the buffy coat, and the sensitivity threshold of trypanosome detection generally ranges from 1 to 20 parasites/mL of blood. For monospecific PCR identification, the most favoured target is minichromosomal nuclear satellite DNA, which has the advantage of a highly repetitive short sequence . Specific satellite primers were used for Trypanozoon, T. vivax, T. congolense savannah-type, T. congolense forest-type [T. congolense forest-type infection using TCF1/TCF2 primers, can be visualized on an agarose gel after staining with ethidium bromide and exposure under ultraviolet light .The virulence of est-type , 22. Theet light . The speet light . Additio3.Trypanosoma congolense forest-type caused severe hypoglycaemia refractory to sugar supplementation. It is possible that the drastic treatment of hypoglycaemia had the paradoxical effect of promoting trypanosome survival. The dog survived but is suspected of being an asymptomatic carrier 6 years after her apparent cure. Further treatment and follow-up should help to clarify and hopefully solve this unclear status.In this dog, infection with


Candidatus Brocadia sinica” and “Ca. Scalindua sp.” in the form of planktonic cells using membrane bioreactors (MBRs). The MBRs were continuously operated for more than 250 d with nitrogen loading rates of 0.48–1.02 and 0.004–0.09 kgN m−3 d−1 for “Ca. Brocadia sinica” and “Ca. Scalindua sp.”, respectively. Planktonic anammox bacterial cells were successfully enriched (>90%) in the MBRs, which was confirmed by fluorescence in-situ hybridization and 16S rRNA gene sequencing analysis. The decay rate and half-saturation constant for NO2− of “Ca. Brocadia sinica” were determined to be 0.0029–0.0081 d−1 and 0.47 mgN L−1, respectively, using enriched planktonic cells. The present study demonstrated that MBR enables the culture of planktonic anammox bacterial cells, which are suitable for studying their ecophysiology and biochemistry.Enrichment cultures of anaerobic ammonium oxidation (anammox) bacteria as planktonic cell suspensions are essential for studying their ecophysiology and biochemistry, while their cultivation is still laborious. The present study aimed to cultivate two phylogenetically distinct anammox bacteria, “ This miomycetes . Activitomycetes , 15, 54.omycetes , 50 and omycetes .2 gas are notorious with these reactors   , in whictiensis” , and morCa. Brocadia sinica” and biofilms of “Ca. Scalindua sp.”, respectively. These biomasses were previously obtained from activated sludge at a Japanese domestic wastewater treatment plant  and 16S rRNA gene sequencing analyses. After planktonic cells were successfully obtained, the decay rate and half-saturation constant for NO2− of “Ca. Brocadia sinica” were determined using enriched planktonic cells.The present study was performed to obtain enrichment cultures of anammox bacteria in the form of planktonic cells using MBRs. Planktonic cells are defined in the present study as cells suspended in cultures as single cells and not settled by gravity sedimentation. For the cultivation of planktonic anammox bacterial cells, two MBRs with a hollow-fiber membrane unit were operated for >250 d after inoculation of granular sludge of “nt plant  and fromnt plant , respect2PO4 (24.4 mg L−1), MgSO4·7H2O (60 mg L−1), CaCl2 (51 mg L−1), yeast extract  (1.0 mg L−1) and 0.5 ml trace element solution I and II (4(SO4)2 and NaNO2 were supplemented into the media at final concentrations of 560–840 and 70–140 mgN L−1 for “Ca. Brocadia sinica” and “Ca. Scalindua sp.”, respectively. An artificial sea salt SEALIFE  was supplemented into the media for “Ca. Scalindua sp.” at a final concentration of 28 g L−1. The detailed mineral composition of the artificial sea salt SEALIFE was described in our previous work  installed with a hollow-fiber membrane unit was used for cultivation of “ sinica” . The memCa. Brocadia sinica was harvested from an up-flow column anammox reactor previously operated in our laboratory (4+ and NO2− (each 10 mM) for more than 1 year. Two hundreds milliliters (ca. 50 g wet) of biomass was collected using a stainless spatula and inoculated into the MBR.As seeding biomass, granular biomass (average diameter of 2–3 mm) of boratory . This coCa. Brocadia sinica”, except for the pore size of the membrane; i.e., 0.4 μm. The culture fluid in the MBR was continuously mixed with a magnetic stirrer at 200 rpm and sparged with 95% Ar-5% CO2 at a flow rate of 10 mL min−1. pH was not controlled but it was in the range of 7.3 to 7.6. The inorganic nutrient medium was fed continuously at 1 L d−1, which resulted in a hydraulic retention time of 2 and 7 d in the 2-L and 10-L glass bottles, respectively. Biomass retention time was not controlled for “Ca. Scalindua sp.”.A 2-L Pyrex screw cap storage glass bottle  was used as the culture vessel. This was changed to a 10-L bottle after 178 d of operation to scale up the cultures. Working volumes of cell cultures were 2 and 7 L in the 2-L and 10-L glass bottle, respectively. A hollow-fiber membrane unit was installed in the glass bottles and operated at 28°C in a dark place . The holca. 1 g wet) taken from the previously operated up-flow column reactor equipped with a non-woven fabric sheet  for NO2− was determined as previously described  and NO2− (14 mgN L−1). Incubation was conducted for 4 h, and culture fluid samples for the determination of NO2− were collected using a disposable plastic syringe every 15 min. Cell growth during incubation was ignored since the incubation time was much shorter than the minimum doubling times of “Ca. Brocadia sinica”   . The valnet) of “Ca. Brocadia sinica” was calculated using planktonic cells. The value of μnet includes specific growth (μ) and decay rates (b) as described in The net specific growth rate . Inhibitory effects of pH, temperature and DO could be ignored because the MBR was operated at pH 7.6–8.6, 37°C and DO at <0.1 mgO L−1, which are favorable conditions for the growth of “Ca. Brocadia sinica”  with an IonPac CS3 cation column and IonPac AS9 anion column  following the instruction manual supplied by the company. Briefly, the gas-sampling tube was connected to a sampling port of the jar-fermentor and the gas sample was continuously collected by sparging Ar gas into the MBR at a flow rate of 10 mL min−1.NConcentrations of total biomass and planktonic cells were determined as protein concentrations. Ten milliliters of enrichment cultures were collected from the MBR into a 10-mL glass test tube  , and 1 ml of the well-mixed culture sample was dispensed into a 1.5 mL centrifuge tube for the determination of total biomass concentration. For planktonic cells, the rest of the enrichment culture was incubated for 5 min without shaking to settle aggregating biomass and then 1 mL supernatant was collected into another 1.5 mL centrifuge tube. The tubes were centrifuged at 14,000 rpm for 10 min, and biomass pellets were suspended in 10% (w/v) sodium dodecyl sulfate solution. After boiling for 15 min, centrifugation was performed at 14,000 rpm for 10 min and the protein concentrations of the supernatant were determined by a DC-protein assay kit  according to the instruction manual.Ca. Scalindua sp.” was determined with a laser diffraction particle size analyzer SALD-2000J .The size distribution of aggregating biomass in enrichment cultures of “Ca. Brocadia sinica” and “Ca. Scalindua sp.” were deposited in the DDBJ nucleotide sequence database under accession numbers AB759554 and AB822932.Sequence data of the partial 16S rRNA gene for “−3 d−1, respectively (4+ and NO2− were simultaneously consumed with NO3− production (2− and consumed NH4+ (ΔNO2− / ΔNH4+) and produced NO3− and consumed NH4+ (ΔNO3− / ΔNH4+) were 1.15–2.12 (average 1.36) and 0–0.22 (average 0.12), respectively. These values are close to the stoichiometric ratios of anammox processes   , 40, ind, <0.01% , 0.03–0., <0.01% .Ca. Brocadia sinica” in the MBR. As shown in Ca. Brocadia sinica” based on the partial 16S rRNA gene sequence determined in the present study (accession number AB759554)  . It shou−1 at 215 d of operation , the concentrations of total biomass and planktonic cells did not change; i.e., 1.31–1.51 and 0.95–1.13 g BSA L−1, respectively  was achieved during the entire operation. In the MBR, both NH4+ and NO2− were simultaneously consumed with NO3− production, and NO2− concentrations in effluents were between 0.05–0.1 mgN L−1 (2− / ΔNH4+ and ΔNO3− / ΔNH4+ were 0.92–1.51 (average 1.13) and 0.01–0.29 (average 0.18), respectively, indicating that anammox was responsible for nitrogen removal in the MBR.Another MBR was operated for the cultivation of “ m−3 d−1 . At 179  mgN L−1 . The stoCa. Scalindua sp.”, previously designated as clone husup-a7 Ca. ScaliCa. Scalindua sp.” were successfully cultivated in the MBR. Although small aggregated biomass (<100 μm) of “Ca. Scalindua sp.” was dominant in the cultures collected at 78 d of operation, planktonic cells dominated at 237 d of operation, as determined by microscopy   ; i.e., 0croscopy , 24, 34.i.e., “Ca. Kuenenia” . These enrichment cultures were obtained using MBRs, indicating that MBR is an essential tool for the cultivation of planktonic anammox bacterial cells. In the present study, enrichment cultures of “Ca. Brocadia sinica” and “Ca. Scalindua sp.” were obtained after 215 and 237 d of operation, respectively. Based on μ of “Ca. Brocadia sinica” , this corresponds to 8–9 cell duplications. As for “Ca. Brocadia sinica”, the exponential growth of planktonic cells initiated after 60 d of operation  supplementation of artificial sea salt and 2) lower nitrogen loading rate and concentrations of NO2− and NH4+ in influents.So far, enrichment cultures of anammox bacteria in the form of planktonic cells have been obtained from three out of five candidate genera; uenenia” , “Ca. Brperation . This laet al. . These conditions were comparable with those employed in the previous study . Specific influences of the above-mentioned cultivation conditions for the proliferation of planktonic anammox bacterial cells are still unclear, and more studies are definitely required to investigate the formation mechanisms of planktonic anammox bacterial cells in relation to cultivation conditions.It should be noted that although some anammox MBRs have been previously constructed for feasibility studies on biological nitrogen removal, granular sludge or small aggregates of anammox bacteria dominated in those MBRs , 46, 53.et al. , they poet al. . On the  surface , which pectively . On the tatively , 13, whitatively . In the us study . Anotherb for “Ca. Brocadia sinica” were determined to be 0.0029–0.0081 d−1 in the present study, which were comparable with those (0.0011 and 0.0048 d−1) previously determined by fitting a mathematical model or batch activity test, respectively    to underCa. Brocadia sinica” in the MBR biomass (>90%) up to 99.9% by Percoll separation (For further improvement of ecophysiological and biochemical studies, pure cultures of anammox bacteria are desired. It was possible to further enrich “paration . This hi