A difference in neuromotor skills was also visible, though only for swing and stance duration, implying a difference in neuromotor development in utero. However, further maturation during the first days after birth does not seem to be affected by intrauterine crowding. We can therefore conclude that L piglets might be considered a smaller and fictitious younger version of N piglets.In polytocous species, such as pigs, the growth of an individual fetus is affected by competition from its littermates and the sow. This intrauterine competition greatly influences postnatal traits such as birth weight and vitality . A lowered vitality is most often observed among low birth weight piglets. Since it has been argued that locomotion might be key to unraveling vitality-related differences, we compared gait development in piglets with a low birth weight and low vitality (L piglets) with piglets with a normal birth weight and normal vitality (N piglets) by means of spatio-temporal gait analysis during locomotion at self-selected speed. Video recordings of L and N piglets walking along a corridor at ten time points (between birth and 96 h after birth) were made and the footfalls were digitized. Hence, self-selected speed, spatio-temporal characteristics and gait symmetry were analyzed to compare motor performance, neuromotor maturation  and gait variability for L and N piglets. The analysis included both absolute and normalized data (according to the dynamic similarity concept), to distinguish neuromotor maturation from effects caused by growth. Results indicate that intrauterine crowding affects locomotion, mainly by impairing growth  With regard to litter size, animals are generally subdivided into monotocous and polytocous species . MonotocThe study of polytocous species is particularly interesting because the growth of an individual fetus is affected by competition from its littermates and the mother. This competition can occur on a systemic , regional (due to the number of fetuses in a particular uterine horn) or local (due to the immediate proximity of neighboring fetuses) level . The effIn pigs, both uterine capacity –15 and pet al. [Needless to say, intrauterine competition greatly influences postnatal traits. In pigs, research has focused mainly on the effect on birth weight (e.g. ), body met al.  argue thAs argued in our previous study , spatio-Our earlier study  showed tMore specifically, we addressed the following questions:self-selected speed (u), stride frequency (f) and stride length (lstride).Is motor performance different for L and N piglets at birth and during early development? We hypothesize a reduced motor performance for L piglets, compared to N piglets, both at birth as during early development. To this end we check for differences in  of u dynamically similar to N piglets after a few hours [u and its components (f and lstride) according to the dynamic similarity principle, generating u’, f’, lstride’ . We. Weu dynIs interlimb coordination at birth and its postnatal development different for L and N piglets? In N piglets interlimb coordination is shown to be innate . Therefotsw’) and stance duration (tst’), duty factor (df), normalized step length (lstep’) and normalized maximum swing height (hswmax’) between L and N piglets.Is intralimb coordination  at birth and its postnatal development different for L piglets, compared to N piglets? We hypothesize this is the case. To this end we compared the development with age of normalized swing  from 11 litters were selected between February and May 2015 on a local farm. The average number of piglets born alive within these litters was 16.5 (± 3.8) . Between 1 and 5 healthy piglets per litter were selected immediately post-partum and ear notched upon selection. Sex was not used as a criterion. In total 16 females and 9 males were selected, for more details on the selected piglets, see et al. [Institutional and national guidelines for the care and use of animals were followed and all experimental procedures involving animals were approved by the Ethical Committee of Animal Experimentation, University of Antwerp, Belgium . Twenty-five domestic piglets ), perpendicular to the camera .et al. [Animals were recorded at ten points in time . The close spacing of these time points allowed for a detailed description and comparison of motor performance, neuromotor maturation and variability of the gait pattern during early development. As explained in Vanden Hole et al.  age 96 het al. [et al. [H), i.e. the distance between the most distal part of the distal phalanx and a fixed point on the pelvis, i.e. the tail base. H was determined for each sequence, in the frame coinciding with midstance of the hind limb closest to the camera.All video sequences were digitized field-by-field using Matlab , following the method described in Vanden Hole et al. . This me [et al. ). For thhswmax) was added to the 19 gait-variables included by Vanden Hole et al. [u, f, lstride and lstep, both absolute and normalized (according to the dynamic similarity principle) variables were studied. For all other spatio-temporal gait variables only the normalized (according to the dynamic similarity principle) variables were studied. The dataset of the N piglets is the same as the one used in Vanden Hole et al. [One extra variable, the clearance of the limb during swing  and leg on the outcome variables, linear mixed models were fitted. Fixed factors included age, category and leg, though the latter was only included if it was relevant for the parameter in question. To avoid the model becoming too complex and because it was not the focus of this study, sex was not included as a covariate. In addition, our previous study showed it to have no significant effect on any of the investigated variables in N piglets  was included as a covariate. Age was included as a repeated measure. To account for the dependence between observations between littermates and within the same animal, random factors were included for sow and piglet (nested in sow), plus random slopes for leg and piglet (nested in sow). Stepwise backwards modelling was again used to simplify the starting model.To evaluate the effect of category on f’, tst’, AIf, AIlstride, AIlstep and AIdf were log transformed, while f, lstride, u’, lstride’, lstep’, tsw’, hswmax’, AItst and AItsw were square root transformed. u, lstep and df required no transformations.To meet normality and/or homoscedasticity assumptions, some outcome variables required transformations. ® Pro 12  was used for the entire analysis. Values were considered statistically significant if p ≤ 0.05. Post hoc analysis with Dunnett’s correction was used to compare the different age groups to CA 96 h, while post hoc analysis with Tukey’s correction was applied for the comparison of different legs.JMPH with age are shown in p < 0.0001). The lack of a significant interaction effect between age and category indicates that the growth pattern was not significantly different for L and N piglets.The change in body mass and H was shorter for L piglets than for N piglets. A fairly gradual increase in H between the age of 0 h and 96 h was visible for both groups (L: 0.11 m (± 0.02) to 0.16 m (± 0.01); N: 0.12 m (± 0.03) to 0.21 m (± 0.03); p < 0.0001). Since body mass remained constant during the first 28 h, the increase of H after 28 h could be attributed to growth, while the increase in H up to the age of 28 h could be attributed to acquiring a more erect posture. As mentioned in Vanden Hole et al. [H via attaining a more erect limb posture took longer, i.e. the entire period between age 0 h and 28 h.Similar to body mass, e et al.  the largu was found at ages 0–8 h, compared to CA 96 h (p < 0.0001 (0 h and 1 h), 0.0013 (2 h), 0.0072 (4 h), 0.0095 (6 h) and 0.0380 (8 h); u was found (0.14 ms-1 (± 0.07)), than for N piglets (0.17 ms-1 (± 0.08), p = 0.0122). Similarly, lstride was shorter for L piglets, compared to N piglets (0.11 m (± 0.04) vs 0.14 m (± 0.05), p = 0.0021; lstride was shorter from age 0 h up to and including 26 h (p < 0.0001 (0 h to 8 h), p = 0.0019 (24 h) and p = 0.0435 (26 h)). L piglets generally showed a shorter lstep compared to N piglets , p = 0.0004, 0.0036, 0.0040 and 0.0422 for 2, 4, 6 and 8 h, respectively), while in N piglets lstep continued to increase for a longer time ). For L piglets we saw an increase in lstep from 0.06 m (± 0.02) to 0.08 m (± 0.03) between the age 0 h and 8h (p < 0.0001 (0 h and 1 h), p = 0.0004 (2 h), p = 0.0036 (4 h), p = 0.0040 (6 h) and p = 0.0422 (8 h)), while N piglets showed an increase from 0.06 m (± 0.04) to 0.12 (± 0.02) between 0 h and 24 h (p < 0.0001 (0 h– 8 h) and p = 0.0210 (24 h). In contrast, no significant effects were found for f . Similarly, lstride’ was not significantly different for L and N piglets (lstride’ was significantly lower at ages 0 to 2 h than at CA 96 h (p < 0.0001 (0 h and 1 h) and 0.0026 (2 h)). lstride’ at ages 0 h, 1 h and 2 h made up 71.59%, 76.21% and 81.40% of lstride’ at CA 96 h, respectively. However, for f’ we did find a lower mean value for L piglets , right front (RF) and right hind (RH) leg the f’ was only significantly different from the f’ at CA 96 h at age 0 h  and p = 0.0010 (RH)).The statistical analysis revealed  piglets . For bot piglets . lstrideThe time lags did not differ significantly between categories and ages. Values for f-lag, h-lag, p-lag (left), p-lag (right), d-lag (LH-RF), d-lag (RH-LF) were 0.51 (± 0.15), 0.49 (± 0.15), 0.68 , 0.34 (± 0.16); 0.26 (± 0.19) and 0.20 (± 0.11), respectively.tsw’ was 24.65% longer for L piglets than for N piglets . For tst’ we also saw an overall difference between L and N piglets (tst’ of L piglets was 13.31% longer than that of N piglets (p = 0.0392). Compared to CA 96 h, only age 0 h showed a significantly longer tst’ , indicating that tst’ reached a stable value already within the first hour after birth. No significant differences between L and N piglets were found for lstep’. lstep’ reached a stable value between the age of 2 h and 4 h, since smaller mean values were observed between ages 0–2 h . For RF the hswmax’ was only different for age 0 h, being 215.79% of the hswmax’ at CA 96 h for L piglets (p = 0.0002) and 188.99% of CA 96 h for N piglets (p < 0.0001). piglets . The tstlegs did . In othef showed a higher value at ages 0 h, 1 h and 2 h, compared to CA 96 h . AIlstride was significantly higher for ages 0 h up to and including 8 h, compared to CA 96 h , 31.32% (± 32.83), 24.11% (± 15.91), 21,27% (± 16.80), 24.40% (± 21.90) and 22.48% (± 22.05), while at CA 96 h AIlstride decreased to 11.56% (± 7.71). Similar results were found for AIlstep, with AIlstep for ages 0, 1, 2, 4, 6 and 8 h showing a significantly higher value than at CA 96 h (p < 0.0001 (ages 0 h and 1 h), p = 0.0010, 0.0040, 0.0006 and 0.0118, respectively; tst was higher at ages 0 h (65.50% (± 31.61)) and 1 h (37.72% (± 34.85)), compared to an AItst of 18.42% (± 15.04) at CA 96 h ) than at CA 96 h (27.13% (± 15.64), p < 0.0001; df was also significantly higher at age 0 h (20.82% (± 16.29)), compared to CA 96 h (11.29% (± 6.65)) , compared to the hind leg pair (22.38% (± 22.94); p = 0.0033). AIlstride also showed a difference between the front and the hind leg pair (lstride of 25.21% (± 27.99) (p = 0.0014). The front leg pair also showed a lower AIlstep (27.82% (± 83.18)) compared to the hind leg pair (28.95% (± 29.98), p = 0.0006, lstride and AIlstep, front legs showed a lower AItst than hind legs  and 12.60% (± 10.08), respectively; Taking a closer look at the leg pairs, we saw differences for AIand AIdf . Howeverleg pair , though u for L and N piglets did not differ significantly, but in fact seemed to form a continuum, with u increasing with a greater body mass  and 0.11 m (± 0.04), respectively), compared to L piglets (0.12 m (± 0.04) and 0.08 m (± 0.03), respectively; p = 0.004 for lstride and p = 0.0277 for lstep). f in function of body mass was different for L and N piglets. For L piglets, body mass had no significant effect on f, i.e. f remained constant. However, for N piglets, we observed a decrease in f as body mass increased .u for L than for N piglets. This lower u can be attributed to a shorter lstride for L piglets, in view of the equal f for both groups. Likely, this difference in motor performance can be attributed to the difference in H (because of size) between both groups. A longer H for N piglets leads to a larger lstride, which in turn leads to a higher u, compared to L piglets (given that L and N piglets show the same f). However, it might be advantageous for L piglets to attain a similar u to N piglets by increasing f in order to compensate for the shorter lstride. A first advantage of increasing their u might be a more balanced competition for teats with N piglets. Selection-related increase in litter size has led to a more severe teat competition [u, is that the chance of being crushed by the sow might be reduced. So then why don’t L piglets increase their f in order to attain a higher u?Consistent with our hypothesis, we see a reduced motor performance for L piglets, indicated by a lower petition . L piglepetition . A seconu’, f’ and lstride’) suggests that muscle recruitment (the fraction of activated fibers) takes place at the same level for both groups. If, however, L piglets were to increase u by means of a higher f, this recruitment level should increase disproportionally because faster walking would not only imply a higher relative load for the muscles, but faster walking would also mean faster contractions, hence less force produced per fiber. As such, the locomotor cost would be influenced directly [u all fibers are already recruited, anyway) L piglet muscles may simply lack the required energy flow to increase f. Piglets, both L and N, are born with low energy reserves. A newborn pig is devoid of brown fat [f remains unaffected in L piglets.The identical motor task of L and N piglets . lstride’, after an initial increase, already stabilizes between 2 h and 4 h after birth. During this short period of increase, neuromotor maturation takes place. f, on the other hand, remains invariable during early development, while f’ shows a short increase during the first hour after birth, due to an increase in H. Interestingly, f’ is lower for L piglets than for N piglet across all ages, though this does not cause an overall difference in u’ for both groups. Contrary to our hypothesis, these results indicate that with regard to motor task, L and N piglets show an identical and equally quick maturation process.The motor task is similar for L and N piglets and stabilizes very quickly, which is indicated by et al. [The lack of changes during early development and between L and N piglets supports our hypothesis based on the results on N piglets , that ilstep’ and hswmax’ are not different for L and N piglets, thus not confirming our hypothesis. In other words the linear dimensions of their step, relative to size, are equal between both groups during early development. In addition, the evolution of both lstep’ and hswmax’ during early development shows the same pattern, which means neuromotor maturation in this regard is the same for L and N piglets.We hypothesized a difference (both at birth and during subsequent development) in individual limb behavior between L and N piglets, but this does not seem to be the case (completely). First off, tst’ and tsw’ are longer for L piglets, compared to N piglets, though afterwards they show the same maturation for both groups. This confirms our hypothesis only partly, since we don’t see a differential maturation between L and N piglets and the difference that is present at birth is maintained throughout early development. These different levels of tst’ and tsw’ at birth indicate a difference in neuromotor development in utero. The prolonged tst’ and tsw’ for L piglets lead to a lower f’, but (as mentioned earlier) not to a lower u’.However, tst’ of less than 1 h. During this first hour the duration of the stance phase is longer than at later ages. In Vanden Hole et al. [hswmax’ might lead to a decreased stability. This is especially visible for the front legs, which, during this first hour after birth show an increase in hswmax’ and tst’. For a more extensive discussion on front versus hind legs, see below.Both L and N piglets show a neuromotor maturation with regard to e et al.  we propotsw’ seems to take longer, with an initial decrease until the age of 4 h, after which (between the age of 4 and 6 h) tsw’ stabilizes at a slightly higher level. This is the same pattern as described for N piglets in Vanden Hole et al. [Neuromotor maturation of the e et al.  though itst’ in itself is longer for L piglets, the tst relative to the duration of the entire stride, is the same for L and N piglets. This is in accordance with results by Biewener [et al. [tsw’. When tsw’ increases, the duration that the front or rear end is only supported by one leg, increases as well. These supporting legs experience an increased amount of force. However, an increase in df reduces the peak ground force that is exerted on a limb during the stance phase [H) is not yet optimal for walking. Afterwards, tsw’ decreases further, in order to walk as energy-efficient as possible [df  is the same for L and N piglets. This shows that, though Biewener , who fou [et al.  who founce phase , 59. Thipossible .et al. [lstride, which seems to take a little longer (between 8 and 24 h) to reach a constant value.Contrary to our hypothesis, L and N piglets show no difference in AIs, indicating the same development and degree of stability in both groups. In accordance with Vanden Hole et al.  the variet al. ) within ts’ and tsw’ between front and hind limbs. Front legs spend a longer time on the ground than hind legs. As a consequence they also exhibit a longer lstep’, which is the displacement of the COM during the stance phase.One cannot discuss gait stability without also considering possible differences among leg pairs. As argued in our previous study  the fronhswmax’ shows a different development for front legs and hind legs. During the first hours piglets lift up their front legs higher than during the rest of the study period. In addition, we see that front legs display a higher degree of symmetry compared to hind legs , a difference that is most pronounced during the first few hours. This makes sense if we consider the front legs to take up an exploratory role. The front limbs are the first to encounter modifications to the environment  and must be the first to adjust their movements [hswmax’ might help with exploring the environment, while a higher degree of symmetry increases stability even if obstacles are encountered.However, for both N and L piglets we see differences between front and hind legs that cannot solely be explained by this difference in body build. ovements . An incru, lstride and lstep of L and N piglets increase in function of a higher body mass. For lstride and lstep we see a lower mean value for L piglets than for N piglets. For u this is not this case, though this is likely due to a smaller number of data points, because u is calculated per piglet and not per leg. For these three variables we can state that L piglets behave as a smaller version of N piglets.The f, we see a different relation with body mass for L and N piglets. In L piglets f remains constant in spite of an increase in body mass, which is in accordance with our earlier proposed theory with regards to the inability of L piglets to adjust their f. However, for N piglets f decreases in function of body mass. This might indicate that N piglets, given that lstride increases because of an increased body mass, lower f to increase the energetic efficiency of walking , leading to a lower u. For u, lstride and lstep we observed an increase in function of body mass, indicating L piglets might be considered a smaller version of N piglets. At birth, the motor task , the linear aspects of limb-specific coordination (lstep’ and hswmax’), the relative limb-phasing and stability of the gait (left-right asymmetry) appear similar for L and N piglets, while we do see different levels of tst’ and tsw’. During the four days after birth, neuromotor maturation shows the same pattern for L and N piglets.We noted a reduced motor performance for L piglets with a lower in utero of L piglets, which after birth negatively affects motor performance. In addition, we see a slight difference in neuromotor skills at birth. This implies a difference in neuromotor development in utero, indicating that L piglets might be considered a fictitious younger version of N piglets. However, it does not seem that intrauterine crowding affects neuromotor maturation after birth.As such, we believe that intrauterine crowding affects locomotion mainly by impairing growth S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.


However, only a minor increment was found for acetate degradation either at 1 g/L or 5 g/L. The stimulatory mechanism of GAC for accelerated syntrophic degradation of propionate and butyrate can be primarily attributed to the triggering effect on acetogenesis, as evidenced by the enrichment of syntrophic bacteria e.g. Thermovirga, Synergistaceae, and Syntrophomonas etc.Granular activated carbon (GAC) could promote methane production from organic wastes, but a wide range of dosages has been reported. In present study, different GAC dosages of 0, 0.5, 5 and 25 g/L were supplemented into anaerobic digesters and the methanogenic degradation kinetics of acetate, propionate and butyrate were characterized, respectively. At high organic load of 5 g/L, the degradation rates of propionate and butyrate increased by 1.5–4.7 and 2.5–7.0 times at varied GAC dosages. The methane production rates ( Meanwhile, two different organic loads of substrate, i.e. 1 g/L and 5 g/L were compared. The rates of VFAs’ degradation and methane generation were evaluated by using first-order kinetics and Modified Gompertz model. The high throughput technique was used for 16s rDNA sequencing to detect the microbial community structure, and the alternation of syntrophic VFAs degrading bacteria and methanogens due to GAC addition was discussed in this study.22.1Inoculum sludge taken from Quyang Sewage Treatment Plant  was pre-cultured in a laboratory scale anaerobic digester. And then the sludge was transferred to three reactors fed with different VFAs, i.e. acetate, propionate and butyrate, respectively, to enrich the specific fatty acids degradation bacteria. After several sequential batches of cultivation, 1 g/L of each VFA species could almost be degraded after 5–7 days. The cultivation temperature was maintained at 35 ± 2 °C.2/g, respectively. Sequentially, the conversion rates of acetate, propionate and butyrate into methane with specific enriched cultures were evaluated in batch studies at the concentration of 1 g/L and 5 g/L. Data was collected after two batches of pre-culture, and each test was carried out in triplicate. The temperature of reactors was maintained at 35 ± 2 °C with an incubator shaker .During experiments, the determined volume of enriched sludge was put into 500 mL serum bottle with 400 mL of digestate liquid to make a final concentration of total volatile suspended solid (TVSS) at 1 g/L. Different dosages of GAC was supplemented to serum bottles i.e. 0, 0.5, 5 and 25 g/L, respectively, which were recorded as GAC0, GAC0.5, GAC5 and GAC25. GAC was purchased from Sinopharm Chemical Reagent CO. LTd. 20–40 mesh GAC was obtained by shive, which apparent density and specific surface area was 430 ± 30 g/L and 875–1185 m4Cl and KH2PO4 were added to the reactors according to the C: N: P = 100:5:1. Additionally, 2 mL/L of the trace element solution was added , the corresponding qualities of NHas added  and the 2.2The volume of methane generation was automatically measured by AMPTS II  equipped with a gas flowmeter. The liquid of each reactor was sampled and analyzed to monitor the variations of total organic carbon (TOC) and VFAs. After filtrated by 0.45 μm filter membrane, the concentration of VFAs was analyzed by high performance liquid chromatography  equipped with refractive index detector. The TOC was analyzed by Total Carbon/Total Nitrogen analyzer .2.3TM DNA isolation kit . Labels of “HAc0”, “HPr0”, “HBu0” stand for the sludge samples taken from GAC0 with respective substrate, and “HAc1”, “HPr1”, “HBu1” stand for the samples taken from GAC5. The microbial community of samples was analyzed by using high-throughput pyrosequencing on an Illumina platform (Illumina Miseq PE300). Amplicon libraries were constructed for pyrosequencing using bacterial primers 515F (50-GTG CCA GCM GCC GCG GTA A-30) and 806R (50-GGA CTA CHVGGG TWT CTA AT-30) for the V4–V5 region of the microbial 16SrRNA gene  at time t, ultimate methane yield (mmol-C/mmol-Cadded) at the end of the incubation, maximum production rate (mmol-C/mmol-Cadded/d) and lag phase (d), respectively for CH4 and CO2 production; e is 2.71828. In Eq. 0C and C(t) is the initial concentration of particular substrate and the concentration at time t; k is the first order degradation constant.Modified Gompertz model Eq.  was fittoduction . The varoduction .(1)MCH4, maximum production rate (maxR), and lag phase (λ) from Modified Gompertz model were presented in CH4P from acetate was 0.45–0.49 mmol-CH4/mmol-Cadded, which was close to the theoretical value of 0.5 mmol-CH4/mmol-Cadded. The average maxR of HAc was 0.16 mmol-CH4/mmol-Cadded/d, which value was slightly higher than the previous study, i.e. 0.107–0.143 mmol-CH4/mmol-Cadded/d .With the initial concentration of 5 g/L, acetate-acclimated culture still showed high metabolic activity and the supplementation of GAC almost did not affect the methane production rate. The ultimate methane yielding However, the methanogenic conversion of propionate and butyrate were obviously inhibited when increasing the substrate concentration from 1 g/L to 5 g/L. The lag-phase time of propionate and butyrate reactors was 4.2 days and 12.7 days for GAC0. It is like previous studies that methanogenesis was vulnerable to high concentration of VFAs, mainly attributing to the inhibition of growth and metabolism of methanogens by undissociated VFAs . Neverthk) of each VFAs species are calculated and presented in 2 is high, only the R2 of HBu (5.0 g/L) was lower than others especially at low GAC dosage. The reason could be attributable to the inhibition effect, leading to the deviation from sigmoidal function. With 5 g/L of propionate as substrate, about 1.5 times of increment was found for the k value in digesters, i.e. from 0.0022 to 0.0056 h−1 when increasing GAC dosage from 0 to 25 g/L. The stimulating effect on degradation of butyrate was more significant, which kinetics value was enhanced by 7.1 times i.e. from 0.0043 to 0.0306 h−1 when GAC dosage increased from 0 to 25 g/L. The above results clearly indicated that the supplementation of GAC could accelerate methanogenesis from propionate and butyrate in a dose-dependent manner. 3O4 might be limited by the supplementing concentration. It should also be noted that although the degradation rates of propionate and butyrate were significantly increased by supplementing GAC, their rates were still lower than that of acetate in the range of 0.0386–0.0431 h−1. These results further confirmed the difficulty in conversion of propionate and butyrate and the importance to further explore the promotion mechanism.The degradation kinetics  have been identified in Aminicenantes species and could be used to convert formate to hydrogen and carbon dioxide as terminal products of fermentation , meanwhile in the propionate- and butyrate-fed reactors, it accounted for 42.8% and 44.8%. Methanosaeta species were predominant with either butyrate or propionate as the substrate. The high abundance of Methanosaeta could be affiliated to the suitable concentration of acetate (max. 68 mM), and it could also consume electrons derived from the oxidation of propionate or butyrate to acetate.On one side, although the selectively enriched archaea varied among reactors with different substrates, reactors b, and thopionate a. In aceMethanosaeta in the digester with GAC generally decreased as compared to the control without GAC. Similar trend was found for Methanobacterium. Whereas the abundance of Methanosarcina and Methanolinea increased. It has been reported that the presence of Methanosarcina could shortly eliminate the negative effect of acidity accumulation and produce methane in high performance  increased in GAC5 than GAC0. Comparatively, the changes on the Archaeal community of in acetate reactors were less than propionate and butyrate reactors, which was in accordance to the similar reaction kinetics of methane generation with varied GAC dosages.On the other side, the relative abundance of formance . To dataduce CH4 . The pro barkeri . In tota3.4.3maxR) and the concentrations of VFAs on the dynamics of microbial community have been analyzed by the representational difference analysis (RDA) and presented in Aminicenantes, Thermovirga, Synergistaceae and Syntrophomonas were also clustered with the same direction of GAC, whereas Methanosaeta was on the contrary coordinate, which is in accordance to the above observation. To data, only Methanosarcina and Methanosaeta are known methanogens to participate in DIET by directly receiving electrons to reduce CO2 and produce CH4  from the biomass attached to GAC. However, in this study, the OTUs clustered to Geobacter detected in sludge with GAC was quite low . Similar result has been previously reported , nevertheless minor increment was found for


Molecular size distribution of meningococcal polysaccharide vaccine is a readily identifiable parameter that directly correlates with the immunogenicity. In this paper, we report a size exclusion chromatography method to determine the molecular size distribution and distribution coefficient value of meningococcal polysaccharide serogroups A, C, W, and Y in meningococcal polysaccharide (ACWY) vaccines. The analyses were performed on a XK16/70 column packed with sepharose CL-4B with six different batches of Ingovax® ACWY, a meningococcal polysaccharide vaccine produced by Incepta Vaccine Ltd., Bangladesh. A quantitative rocket immunoelectrophoresis assay was employed to determine the polysaccharide contents of each serogroup. The calculated distribution coefficient values of serogroups A, C, W, and Y were found to be 0.26 ± 0.16, 0.21 ± 0.11, 0.21 ± 0.11, and 0.14 ± 0.12, respectively, and met the requirements of British Pharmacopeia. The method was proved to be robust for determining the distribution coefficient values which is an obligatory requirement for vaccine lot release. Neisseria meningitidis is the prime cause of bacterial meningitis and fatal sepsis in human [ Neisseria meningitidis group A or C infection in Bangladesh. It was also claimed that the case-fatality ratio for these meningococcal infections was 10%.in human , 2. It hin human , 4. Onlyin human . In mostin human . The relin human –9. In Afin human . Serogroin human –13. In rin human . India, in human . In Bangin human , 17. It  N. meningitidis are immunogenic and used as vaccine against meningococcal diseases. The meningococcal polysaccharide ACWY (PS) vaccine is derived from capsular polysaccharides of meningococci group A , C , W , and Y  [Capsular polysaccharides ofc bonds) , 19. In c bonds) –22. Thisc bonds) . Moreovec bonds) –22.The idea of molecular size based separation by chromatography, also known as size exclusion chromatography (SEC), was first introduced by Synge . It is aMeningococcal polysaccharide can be estimated by assaying either phosphorus content (for serogroup A) or sialic acid content  , 30. HowKD) value of each serogroup of six different batches of meningococcal PS vaccines.Analysis of MSD of individual polysaccharide in multivalent vaccine is always challenging. A rapid and reproducible method for estimation of MSD of meningococcal tetravalent vaccine of ACWY may help vaccine researcher from academy as well as industry to develop effective multivalent vaccines. In the present investigation, we described a SEC method for determination of MSD of tetravalent meningococcal polysaccharide vaccine of ACWY. This experiment mainly focused on calculation and comparative analysis of distribution coefficient  for analysis.Ingovax ACWY, a tetravalent meningococcal polysaccharide vaccine produced by Incepta Vaccine Ltd., Bangladesh, was used in this study. It is a freeze-dried vaccine containing ≥50 μm) was used in this study. The column packed with sepharose CL-4B (particle size 45 μm–165 μm) . These cross-linked bead formed agarose matrix particles have an exclusion limit between 70 and 20,000 KDa. All the chromatography operation was conducted at temperature of 23 ± 2°C.XK16/70  column (porosity 10 μL of 2% (v/v) acetone (Sigma) through the column using purified water as eluent. By Unicorn software the packed column theoretical plate number was calculated as 7173 plates/m and peak asymmetry factor was found 1.05 which matches the acceptance criteria of sepharose CL-4B for column use . The Unicorn 5.2 software package was used for programming method run and data analysis. The column performance test was conducted by injecting 200 lumn use .The column was equilibrated with 1.5 column volume (CV) equal to 187.5 mL (1 CV = 125 mL) of 0.2 M NaCl . 2 mL of reconstituted vaccine samples (using 2 mL sample loop) was injected and run with a flow rate of 0.3 mL/min. The automatic fraction collector was applied to collect fixed volume of 2 mL fractions. The fractions were stored at −20°C until further test.KD values for Ingovax ACWY vaccines were calculated according to Ackers' formula [Vo is void volume, Vt is total bed volume, and Ve is elution volume.The  formula , 32,(1)KVo interprets the exterior part of the beads and is determined by using a molecule which is larger than the exclusion limit of a particular matrix. Small molecule elutes at the end of the column and is determined as Vt. In this method, Vo and Vt were determined by 0.2% (w/v) blue dextran 2000, a polysaccharide covalently bonded with blue dye, MW 2000 KDa , and 0.5% (w/v) sodium azide (MW 65 Da) (Sigma), respectively. The absorbances for blue dextran 2000 and sodium azide were monitored at 206 nm and 260 nm, respectively (Ve) was calculated by estimating the principal peak of polysaccharides. In this experiment, we have also calculated the recovered percentage of eluted polysaccharide before KD value 0.50 by following formula:Here, ectively . The eluμL of Neisseria meningitidis Antiserum (BD) was added and mixed gently. The gel was immediately poured onto a gel tray placed on a horizontal surface. The gel was allowed to solidify and 3 mm sample wells were made using gel-puncher. The gel was set in the electrophoresis unit. The reference standard  solution was 100 μg/mL of polysaccharide in 0.1 M Tris buffer, pH 8.2. The total amounts of samples were distributed in 8 different pools for our experiment suitability. 8 μL of samples and standards were directly loaded to the well in duplicate. Electrophoretic run was conducted for 15 hr at 9 mA. The gel was stained by staining solution containing Coomassie brilliant blue 0.1% (w/v), methanol 45% (v/v), and glacial acetic acid 10% (v/v) for 40 minutes using Mini-Orbital Shaker with gentle agitation, and destained in destaining solution containing acetic acid 7% (v/v) and methanol 10% (v/v).Rocket immunoelectrophoresis (RIE) was used for determination of polysaccharide content in each serogroup. Briefly, agarose (1%) (HiMedia) was melted in 20 mL of 0.1 M Tris buffer, pH 8.2 by boiling in heating mantle. Liquid agarose was cooled to 50–55°C and 200 The polysaccharide content was calculated by following formula:KD value of meningococcal polysaccharide serogroups A, C, W, and Y in meningococcal PS vaccine. For this purpose, Vo and Vt were determined by blue dextran 2000 and sodium azide, respectively. http://dx.doi.org/10.1155/2016/9404068). The chromatogram indicated that the peak started from 35 mL in all six batches . In case of serogroups W and Y, the principal peak was at C5 (Ve = 58 mL) and B7 (Ve = 38 mL), respectively. The KD values for serogroups A, C, W, and Y were calculated using Ackers' formula [Ve = 74 mL) at batch 15002. For serogroups C, W, and Y, the principal peak was found at the same fraction C7 (Ve = 62 mL). The KD value for serogroups C, W, and Y was 0.30 while it was 0.43 for serogroup A at this batch. The elution patterns of batches 15001, 15002, 15003, and 15005 were indistinguishable from each other. However, it was slightly different at batches 15004 and 15006. Interestingly, all the principal peaks appeared at the same position for each serogroup at batches 15004  and 15006 , though the elution patterns were found to be different. The KD values were 0.03 for batch 15004 and 0.12 for batch 15006. In the cases of batches 15001, 15003, and 15005, the principal peak appeared at fraction C5 (Ve = 58 mL) for both serogroups C and W while the KD value was found to be 0.26. Besides, for serogroup A the principal peak was seen at fraction C10 (Ve = 68 mL) with KD value of 0.37 at batches 15001 and 15003. The mean KD ± SD of serogroups A, C, W, and Y, derived from the analysis of six different batches, corresponded to 0.26 ± 0.16, 0.21 ± 0.11, 0.21 ± 0.11, and 0.14 ± 0.12, respectively.At batch 15001, the principle peak of meningococcal serogroup A was observed at fraction C10 with  formula , 32 and KD value 0.5 for each serogroup of six batches. The mean ± SD recovery percentages of A, C, W, and Y were 90.78 ± 8.11, 91.50 ± 5.89, 86.64 ± 6.42, and 84.05 ± 2.74, respectively. The lowest recovery of serogroups A, C, and W was found in batch 15004  and in case of Y it was found in batch 15001 (81.16%)  . Each ofThe MSD is a key indicator for process consistency, confirming the vaccines immunogenicity and safety –37. TherKD values of six batches of Ingovax ACWY vaccine and analysed the batch-to-batch fluctuations. The lower KD value indicates the higher molecular size of polysaccharide which suggests the polysaccharide to be more immunogenic [KD values of individual serogroups of six batches were compared where lowest values were found in all serogroups at batches 15004 and 15006 while the highest values were found at batch 15002. This can be explained from the fact that determination of absolute molecular size distribution is difficult as a result of polydispersity ranging from 5 × 104 to 2 × 105 Da [KD values could be considered insignificant. Misplay of formulation and filling may affect the structural integrity and size of polysaccharides [We identified unogenic , 38. In × 105 Da . Therefocharides  and hydrcharides , 39. In KD value for the principal peak should not be greater than 0.70 for serogroups A and C; 0.57 for serogroup Y; and 0.68 for serogroup W for meningococcal PS vaccine. In our study, the highest values of serogroups A, C, W, and Y were found to be 0.43, 0.30, 0.30, and 0.30, respectively in batch 15002. Therefore, the KD values of each serogroup of six batches of Ingovax ACWY vaccine comply with the requirements of BP [KD value 0.50. In this study, it is found that all the study batches of Ingovax ACWY also meet the requirements of BP [According to the requirements of BP , the KDts of BP . Additiots of BP , at leasts of BP  for recots of BP  and WHO ts of BP .KD values of different serogroups of meningococcal polysaccharide vaccine, as an effective means of indicating reproducibility, accuracy, and performance at industrial settings.SEC could be employed successfully for MSD analysis of polysaccharide vaccine and meaningful information in regard to reproducibility and accuracy of a method could be extracted towards fulfilling the requirements of regulatory authority's approval of a vaccine for human use. The present study reports for the first time the use of SEC on a XK16/70 column packed with sepharose CL-4B for the determination of S1: The polysaccharide content of all fractions was calculated from the value of rocket height of samples & standards and standard's concentrations. S2: The total percentage of recovered polysaccharide for all serogroups was shown.


Listeria monocytogenes has been widely investigated in the poultry production chain from the processing plant to the final product. However, limited data are available on Listeria species, including Listeria monocytogenes, in the poultry farm environment. Therefore, fecal and soil samples from 37 pastured poultry flocks from 10 all-natural farms over 3 years were assessed to determine the prevalence and diversity of Listeria within these alternative poultry farm environments using standard cultural and molecular methods. Listeria species were isolated in 15% of poultry farm samples and included Listeria innocua (65.7%), L. monocytogenes (17.4%), and Listeria welshimeri (15.1%). Additional multiplex PCR serotyping showed group 1/2a-3a to be the most dominant L. monocytogenes serovar group. Based on these results, monoculture growth experiments were conducted on four Listeria soil isolates (three L. monocytogenes isolates representing the three recovered serovar groups and one L. innocua isolate) to determine if culture medium [tripticase soy broth (TSB) and University of Vermont modified Listeria enrichment broth (UVM)], inoculum concentration (102 or 105 CFU/ml), or incubation temperature  differentially affected these Listeria species. Overall, very few significant growth differences were observed between the behavior of the three L. monocytogenes isolates (representing the three recovered serovar groups) under the growth conditions tested. Alternatively, at 30°C in UVM with the lower inoculum concentration, the L. innocua isolate had a significantly shorter lag phase than the L. monocytogenes isolates. In coculture growth studies under these same incubation conditions, the lag phase of L. innocua and L. monocytogenes was similar, but the final concentration of L. innocua was significantly higher than L. monocytogenes. However, cocultures in UVM for high inoculum concentration did not show preferential growth of L. innocua over L. monocytogenes. These results indicate that the use of UVM as an enrichment medium may preferentially allow L. innocua to outcompete L. monocytogenes at low concentrations, biasing the Listeria prevalence from these farm samples toward L. innocua and potentially underreporting the presence of L. monocytogenes in these environments.The occurrence of  Listeria is currently comprised of 17 species, including 11 Listeria species described since 2009 , taking into account that it could be a potential source of this pathogen into food processing plants. Few studies have investigated and characterized Listeria species in the farm environment, with L. innocua being the predominant species found on grow-out farms, representing ≤78% of all isolated Listeria species  during live production on pastured poultry farms and (2) evaluate whether the distribution of recovered Listeria spp. could be explained by a differential growth in the enrichment broth used in this study, or accurately reflected the native species distribution in the environment.While commercial, conventional production represents the majority of the U.S. poultry market, alternative production systems  are becoming more prevalent and there is very limited information related to the prevalence of ironment . TherefoTen farms within the southeastern United States were sampled over a period of 3 years from 2014 to 2016), representing 37 pasture-raised broiler flocks. Farm descriptions are available in Table 4 to 2016Listeria from these environmental samples were performed using a modified version of the USDA-FSIS MLG 8.10 method  and Fraser Broth , both requiring overnight incubation for 24 h at 30°C. One loopful of the Fraser’s enrichment culture was streaked on Listeria selective agar  for the isolation of Listeria colonies. These plates were incubated overnight at 30°C, and on each plate three Listeria-like colonies per positive samples were picked and kept for further identification tests. Stock cultures were prepared by growing Listeria strains in tripticase soy broth  at 37°C. After washing in sterile water, the cell pellet was suspended in a brain heart infusion (BHI) broth  with 25% of glycerol, aliquoted (300 µl in microtubes) and frozen at −80°C until further utilization.Enrichment and isolation of 0 method . Three gListeria colonies recovered on LSA were determined by multiplex PCR , 1 µM of each livN, Igr, and lin2 reverse and forward primers (for Pool 1) or 1 µM of each lwe, Lmo, and lse reverse and forward primers (for Pool 2) and quantity sufficient (qs) of water. For negative controls, sterile water was added instead of template DNA. The cycling program consisted of 1 cycle at 95°C for 9 min; 30 cycles at 94°C for 30 s, at 60°C for 30 s, and at 72°C for 1 min; and 1 cycle at 72°C for 7 min. The serovar group of isolates classified as L. monocytogenes was determined by multiplex PCR using five sets of primers , 1 µM of each Lmo0737, ORF2819, and ORF2110 reverse and forward primers, 1.5 µM of Lmo1118 reverse and forward primers, 0.2 µM of prs reverse and forward primers and qs water. For negative controls, sterile water was added instead of template DNA. PCR was performed with an initial denaturation step at 94°C for 3 min; 35 cycles of 94°C for 0.40 min, 53°C for 1.15 min and 72°C for 1.15 min; and 1 final cycle for 72°C for 7 min. PCR reactions were performed in an Eppendorf Mastercycler EP Gradient S (Eppendorf). After the completion of all cycles, 18 µl of PCR product was mixed with 3 µl of BlueJuice™ loading buffer  and separated on a 2% E-gel® with SYBR-safe™ (Invitrogen) along with 12 µl of E-Gel™ 1 kb Plus DNA Ladder (Invitrogen).The species of presumptive plex PCR . In shor primers . BrieflyListeria species found from farm distribution data, three L. monocytogenes strains representing the different recovered serovar groups  and one L. innocua were selected for the growth experiments. Pre-cultures were prepared by inoculating 60 ml of TSB with 100 µl of the thawed stock culture and incubated for 24 h at 30°C while shaking (150 rpm). After 24 h, cell density was estimated spectrophotometrically by measuring the optical density (OD) at 600 nm (OD600nm) with the Thermo Scientific Spectronic 200™ (Fisher Scientific). Pre-cultures were initially diluted in TSB or UVM to a concentration of 106 CFU/ml, and then serially diluted in TSB or UVM to obtain final inoculum concentrations of 105 and 102 CFU/ml.Based on the two main 5 and 102 CFU/ml) for the four Listeria strains was aliquoted into wells of a microplate , with five repeats of each culture condition (strain × medium × concentration) per plate. Negative controls consisted on 0.4 ml of uninoculated TSB and UVM (five repeats) incubated along the cultures. For each culture, two independent plate repeats containing all treatment combinations were performed. The inoculated microplate was placed in a Bioscreen C microbiology reader , which was operated by a computer with Growth Curves Software, v 2.28 . The microbiology reader recorded the OD values of cultures at 20-min intervals after a plate shaking of 10 s at a medium speed (30 shakes/min). Three incubation temperatures were chosen, and the corresponding incubation times were adjusted to make sure that all growth curves reach the stationary phase by the end of the experiment. Plates were incubated at (i) 20°C, estimated soil temperature calculated upon the average of the atmospheric temperatures encountered during the sampling period (from March to August), for 48 h, (ii) 30°C, recommended temperature used for the enrichment procedure of Listeria spp. in UVM medium, for 24 h, and (iii) 42°C, expected temperature inside the chicken intestine, for 24 h.A volume of 0.4 ml of each culture . Three different coculture mixtures were used to observe coculture growth effects: (1) L. monocytogenes 1/2a-3a to L. innocua ratio of 102:102 CFU/ml in UVM, (2) L. monocytogenes 1/2a-3a to L. innocua ratio of 105:105 CFU/ml in UVM, and (3) L. monocytogenes 1/2a-3a to L. innocua ratio of 102:102 CFU/ml in TSB. As positive controls, monocultures of L. monocytogenes 1/2a-3a and L. innocua were tested under the same conditions (medium × concentration) as the coculture mixtures, while negative controls consisted of uninoculated TSB or UVM, respectively. Each coculture mixture was inoculated individually into a microplate along with positive and negative controls with a final inoculum volume of 0.4 ml for each condition. The OD was recorded at 30-min intervals after a brief plate shaking of 10 s at a medium speed (30 shakes/min) during the incubation at 30°C for 24 h. To quantify the growth of L. monocytogenes 1/2a-3a and L. innocua in coculture, 100 µl aliquots were sampled every hour from the microwell plate and serially 10-fold diluted. Appropriate dilutions were plated on Rapid’L.mono medium . Blue and white colonies were enumerated as L. monocytogenes 1/2a-3a and L. innocua, respectively.Since no significant growth differences were observed among the three y is the OD value measured, t is the time (h), μmax is the maximum specific growth rate (h−1), A is the maximum OD value attained, and λ is the lag time (h). Within the m-file written in Matlab, the lsqcurvefit function (a nonlinear least-squares solver for data fitting) was utilized to fit the growth curves by first using the following Gompertz equation:Growth curves were plotted based on OD values over time. Each bacterial growth curve was fitted to a modified Gompertz model using Matlab 2007b. The model equation is as follows:A, B, and C terms were used to determine the growth parameters of interest in the model equation as follows:Then, the R2 value (coefficient of determination) for each resulting fit was calculated. A four-way analysis of variance (ANOVA) was performed separately on each growth parameter , followed by Tukey’s post hoc test in R software v3.2.1. Factors included in the model were the Listeria strains, the culture medium, the inoculum concentration, and the incubation temperature. For the coculture experiments, μmax and stationary phase cell densities  were log10-transformed before ANOVA, and a Tukey’s post hoc test was used to group treatments. For all analyses, differences among groups were considered significant if p ≤ 0.05.The raw data for each growth curve were graphed along with the resulting fit, and the Listeria species varied according to the sampling year , which is in the range of Listeria species prevalences reported in poultry-related environmental samples (from 1.4 to 53%) such as broiler litter, farm feed, farm drinking water, soil, and grass (L. innocua (65.7%), L. monocytogenes (17.4%), and L. welshimeri (15.1%), and each of these species were recovered from at least half of the broiler farms , and all three species were recovered from soil samples in all 3 years of the study  were collected from 37 flocks on 10 pastured poultry farms over a 3-year period, and the distribution of nd grass , 39, 40 nd grass , 25. In ironment –26, 40, frequent , 40, mosytogenes –43.Listeria monocytogenes was isolated from 5.8, 0.3, and 1.0% of all samples collected in 2014, 2015, and 2016, respectively, with 87% (26/30) recovered during 2014 , 1/2b-3b-7 (20%), and 4b-4d-4e (10%). Interestingly, over the 3-year sampling period, only one L. monocytogenes-positive flock  harbored more than one serotype, demonstrating the potential clonal nature of L. monocytogenes within a flock or on a farm  and pets present on the production site . These ay flocks , 46. Thiisolates . Anotherion site .L. innocua compared with L. monocytogenes on pastured poultry grow-out farms, which has been supported by other studies reporting the incidence and characterization of Listeria species in the commercial poultry farm environment , monoculture and coculture growth studies were performed. Three L. monocytogenes isolates  and one L. innocua isolate were selected to compare their growth capacity in liquid media. Bacterial growth was monitored by recording the OD of a culture in growth media (TSB and UVM) inoculated at different initial concentrations (102 and 105 CFU/ml) and incubated at 20°C , 30°C (UVM enrichment temperature according to USDA-FSIS MLG 8.10), or 42°C (broiler body temperature). Curve modeling was performed with the Gompertz function that fits the data with R2 values ranging from 0.674 to 0.998, indicating a good fit. From the modified Gompertz equation, three relevant parameters  were determined for each curve and subsequently used to statistically compare the bacterial growth of the Listeria strains under the different cultural conditions. Using a four-way ANOVA , we investigated whether the culture medium, the inoculum concentration, and the incubation temperature could explain the global variation observed between the growth curves. In the same model, we more specifically examine the growth differences between the four Listeria isolates for a single culture condition. The parameters λ, μmax, and ODmax representing bacterial growth characteristics were used in the model.Our field results showed a higher prevalence of ironment , 24, 26.F = 801, p < 0.0001; Figure F = 174, p < 0.0001) in both TSB and UVM enrichment media for all Listeria strains, as has been showed in other growth media for both L. monocytogenes and L. innocua . While lag time was significantly shorter in TSB compared with UVM, this was the weakest association of the major growth variables tested .Unsurprisingly, λ was significantly shorter for the higher inoculum concentrations for all strains, enrichment media, and incubation temperatures , where the four Listeria strains grew faster in TSB than UVM  than Listeria enrichment media  . This is in agreement with Duh and Schaffner .While many of the growth variables tested significantly effected μ-Fraser) , 33. We chaffner , who shoth media . The gromax, the enrichment medium was the dominant growth variable affecting ODmax  with significantly higher maximum optical densities found in the treatments grown in TSB . Unlike λ or μmax, initial inoculum concentrations did not have a significant effect of ODmax overall , although limited effects were observed at treatments incubated at 42°C.As was observed for μB Figure , 54. ForListeria spp. overall, the question of the differential effect between specific Listeria species still remained. While there were some exceptions, generally there were no significant differences between the three L. monocytogenes strains in terms of λ, μmax, or ODmax, and regardless of enrichment media, the L. innocua strain was unable to grow at broiler body temperature (42°C) when the initial inoculum concentrations was 102 CFU/ml (TSBLow and UVMLow). Significant differences in lag time between the three L. monocytogenes strains and the L. innocua strain varied based on the growth variables , where there were no significant differences between the four Listeria strains , and Half-Fraser (incubated at 37°C) enrichment media  for any of the growth variables, with the significant differences found at 42°C  we found that L. innocua exhibited a significantly shorter lag time than the L. monocytogenes strains in monocultures, especially for low initial inoculum concentrations (102 CFU/ml). To determine if L. innocua has any direct competitive growth advantages over L. monocytogenes in UVM, coculture experiments were performed using the L. innocua strain and the L. monocytogenes 1/2a-3a strain . When both strains were inoculated into the coculture at 105 CFU/ml , although L. monocytogenes densities did begin to exceed L. innocua cell densities after 24 h. When both strains started at the lower inoculum level (102 CFU/ml), while λ and μmax were similar, L. innocua reached a significantly higher stationary phase cell density compared with L. monocytogenes , with L. monocytogenes cell densities being ~3× greater than L. innocua  compared with UVM (6.38 ± 0.05 log10 CFU/ml) under those growth conditions , L. innocua had a significantly shorter lag phase (monoculture) and a significantly higher stationary phase cell density (coculture) compared with L. monocytogenes; these growth advantages occurred at low initial inoculum concentrations simulating the low levels of Listeria species encountered in the environment. Based on these results, it is possible that UVM enrichment medium either preferentially supports L. innocua growth over L. monocytogenes, or preferentially restricts L. monocytogenes growth, and that this enrichment step may be biasing the recovery of L. innocua over L. monocytogenes from live production samples. Considering the public health importance of accurately identifying the source of L. monocytogenes outbreaks, future work will need to understand the cultural and molecular mechanisms of this preferential L. innocua growth in UVM, and alternative enrichment methods for L. monocytogenes may need to be considered.In our study, we found that AL and MR helped to develop experiments, analyze data, and prepare manuscript. ML helped in data analysis and manuscript preparation.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. The reviewer KG declared a shared affiliation, with no collaboration, with the authors to the handling editor.


Trace and toxic elements in these samples were determined by using inductively coupled plasma-atomic emission spectrometry (ICP-OES) following acid digestion. The order of decreasing mean metal concentrations in raw materials (mg/kg) was found to be as follows: Fe (440) > Mn (162) > Zn (45.8) > Cu (12) > Pb (10.4) > Ni (5.4) > Cr (2.9) > Co (0.91) > Cd (0.5). It was found that 21% of the analyzed samples contain both Cd and Pb above their permissible limits. However, the infusions produced from these plants were found to contain fewer amounts of metals than the raw materials. Studied metals had the following mass transfer percentages to the infusion solutions . The estimated daily intake calculated for the toxic elements Pb and Cd through the consumption of herbal infusions was far below the tolerable daily intake set by the World Health Organization (WHO). Medicinal plants and their various products  are well known as herbal medicines; they have been available for many hundreds of years for treatment of human diseases. Nowadays, various species of herbs are widely used as raw materials in the pharmaceutical industries and also extensively consumed as home remedies . RecentlThe increasing popularity of herbal products as medications has produced fear about their quality and safety for human consumption. These products may be contaminated with microbial contaminants, chlorinated pesticides, heavy metals, and other chemical toxins , 8. The μg/kg, 184–256 μg/kg, and 288–1625 μg/kg, respectively [μg/kg and 143–268 μg/kg, respectively, whereas Pb was found at high elevated concentrations with a range of 6452–8633 μg/kg [The growing condition of plant, chemical treatments, type of plant species, processing steps, and storage condition are important factors affecting the levels of different metals in herbal preparations . Previouectively . However33 μg/kg . The her−1 was used. All glassware used in the present study was previously soaked in 10% (v/v) HNO3 solution for 12 h and rinsed with deionized water. Nitric acid  and hydrogen peroxide  were of ultrapure quality . Standard calibration of mixed and individual metal solutions of Fe, Zn, Cu, Cr, Ni, Mn, Co, Pb, and Cd  at concentration of 1000 mg L−1 was appropriately diluted and used to calibrate the ICP-OES before metal determination. A certified reference materials (NIST 1570a), spinach leave, obtained from National Institute of Standards and Technology, USA, was used for method validation.All solutions were prepared with analytical reagent grade chemicals. Deionized water with specific resistivity of 18.0 M Ω cmA total of 54 samples of various commercially available medicinal herbs were randomly purchased from general stores in Irbid city, Jordan. The herbal samples were commercially available in the form of tea bags packaged in boxes with 12–24 pieces of tea bags; each bag contains 1.1–2.7 g of the herbs raw material. The raw herbal material from all tea bags in one box was placed in polyethylene container, mixed, homogenized, and stored at room temperature. Brand codes, contents, and other information are presented in 3 (69%)–H2O2 (30%). Each beaker was covered with a watch glass and heated on hot plate at 150–180°C; aliquots of nitric acid were added until the solutions were clear. Solutions were contentiously boiled until the volume for each sample reduced to about 5 ml. The solutions were then allowed to cool, filtered (glass wool), and diluted up to 50 ml with acidified (HNO3) deionized water and then placed in acid washed 60 ml polyethylene bottles.Aliquots (1-2 g) of the herbal raw material were accurately weighed into a precleaned glass beaker and digested with 15 ml of a mixture of freshly prepared solution of (1 : 1) (v/v) HNO3 and evaporated to 100 ml.The herbal infusions were prepared by adding 200 ml of hot (97°C) Milli-Q water to a weighed portion (2 g) of plant material. The suspension was then stirred with a glass-rod stirrer for 1 minute and steeped for a total of 10 min, as recommended by the manufacturers. The 10-minute infusion time was found to be adequate to prepare a good quality tea beverage . SubsequAll digested samples and infusions were analyzed, in triplicate, for metal contents using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) (VISTA-MPX instrument). The simultaneous ICP-OES was equipped with axial vision, a radio frequency (RF) source of 30 MHz, a CCD (Charge Coupled Device), a peristaltic pump, and a glass concentric nebulizer.In order to validate the instrumental methods and analytical procedures for accuracy, all the samples were taken in triplicate and all measurements were run in triplicate for standards and samples. Analytical blank involving all reagents was run (in triplicate) to check for interferences and cross-contamination of every batch of 15 samples. Certified reference material (NIST 1570a) was also analyzed every batch. Accuracy was determined by comparing the measured concentrations with the certified values and was expressed as a percentage recovery (% recov.). The achieved results were in good agreement with certified values. The results are given in The concentrations of Fe, Cu, Zn, Mn, Cr, Co, Ni, Cd, and Pb elements in 54 samples of individuals and mixtures of medicinal herbal plants were determined by using ICP-OES. The mean metals concentrations in raw herbal materials are listed in R-square less than 0.004), while Mn has a strong correlation (R-square 0.90). The metals Zn, Co, and Cu had good correlations with R-square values of 0.67, 0.64, and 0.5 respectively. Weak correlations were observed for Ni, Pb, and Cd with R-square values near 0.34.The results for the mean concentrations of studied metals in herbal infusions are presented in The extraction efficiency of metals from the herbal raw material to the water infusion was found to be influenced by the kind of herbal species, while Fe extraction efficiency was below 10% in most cases, it reaches more than 25% for ginger, cumin, and fennel flower. The extraction efficiencies for Zn, Cd, and Pb were high for roselle and exceed 60%. Thyme infusion was found to contain 50% of the initial chromium amount.The average iron content of the medicinal herbs was in the range of 6.2 mg/kg to 1477 mg/kg with the lowest value in cumin and highest value in peppermint. Samples of chamomile, roots of shirsh zallouh, thyme, and sage had high iron concentrations of 824 mg/kg, 686 mg/kg, 556 mg/kg, and 547 mg/kg, respectively. The mean iron content of all the analyzed samples was 440 mg/kg. In another related work, chamomile and sage had comparable iron contents of 502.7 mg/kg and 297.4 mg/kg, respectively . SimilarManganese mean concentration was 162 mg/kg with a range of 1.8 mg/kg (oliban) to 715 mg/kg (roselle). In addition to roselle; the thyme, cinnamon, ginger, and chamomile samples were found to contain significant amounts of manganese as 324 mg/kg, 251 mg/kg, 250 mg/kg, and 185 mg/kg, respectively. Similar concentration range of manganese (<6 mg/kg to 450.7 mg/kg) was reported for some medicinal plants . HoweverZinc and cooper were high in roselle (141 mg/kg) and peppermint 23.7 mg/kg), respectively. The mean zinc and copper concentrations in all of the analyzed herbal samples were 45.8 mg/kg and 12.0 mg/kg, respectively. Zinc concentrations varied from 4.6 mg/kg to 141 mg/kg and copper concentrations ranged from 3.37 mg/kg to 23.7 mg/kg. In a related work, similar concentrations ranges of zinc and copper were reported as 3.75–88 mg/kg and 3.32–30.2 mg/kg, respectively . In a re.7 mg/kg,Lead and cadmium are toxic elements for human; they perform no beneficial biological roles and can be very dangerous even at low concentrations. The highest mean concentration of lead in individual herbs was detected in thyme (19.3 mg/kg) followed by oliban 17.7 mg/kg), roselle (17.4 mg/kg), and peppermint 16.2 mg/kg). Among the mixtures of herbs, the Chinese herbal mixture for slimming was found to contain a high mean concentration of lead (28.7 mg/kg). The lowest mean concentration of lead was observed in fennel flower (black seed) and fenugreek where it was below the detection limit (0.06 mg/kg). The mean concentration of lead in all of the analyzed samples was 10.4 mg/kg. Comparable result for the lead concentration range has recently been reported as from below detection limit to 33.8 mg/kg . In rela.2 mg/kg..7 mg/kg,The content of cadmium in the analyzed herbs varied between <0.04 mg/kg and 1.27 mg/kg with an average value of 0.5 mg/kg. The highest cadmium content was found in allergic asthma mixture (mix 3) as 1.27 mg/kg and Chinese herbal mixture (mix 8) as 1.25 mg/kg. In the individual herbs, cadmium was detected at high concentrations in roselle (1.01 mg/kg), thyme (0.92 mg/kg), lemon balm (0.69 mg/kg), and sage (0.65 mg/kg). Comparing the results obtained in this research with results found in the literature, the cadmium concentration range is similar to that reported by Onwordil et al. for various types of herbal medicines (0.48–3.08 mg/kg) , the resThe concentration ranges of nickel and chromium are similar; they were measured as from 0.53 mg/kg to 15.7 mg/kg and from <0.03 to 9.43, respectively. The mean nickel and chromium concentrations in all of the analyzed herbal samples were 5.44 mg/kg and 2.9 mg/kg, respectively. In related work, a concentration range of 0.72 mg/kg–13.1 mg/kg with an average of 3.62 mg/kg and a concentration range 0.44 to 8.71 with an average of 2.5 have been reported for nickel and chromium, respectively . SimilarCobalt is the least detected element in the analyzed herbs; it was quantified in 54% of the samples in the range of <0.03 mg/kg to 5.67 mg/kg with mean value of 0.91 mg/kg. High mean concentrations of cobalt were detected in roselle (5.19 mg/kg), sage (1.83 mg/kg), anise (1.6 mg/kg), and peppermint (1.02 mg/kg). In the herbal mixture, high cobalt concentrations were found in allergic asthma mixture (mix 3) (3.92 mg/kg) and coughing mixture (mix 3) (3.59 mg/kg). Comparable results for the cobalt concentration ranges in herbal medicines have been reported as 0.05–2.35 mg/kg , 0.08–3.The extraction efficiencies of various metals from raw material to the water infusions were found to be variable; this was also observed by other related studies. For instance, Queralt et al.  showed tμg/day and 0.28 μg/day for Pb and Cd, respectively. These values were far below the tolerable daily intake of Pb (0.3 mg/day) and Cd (1.5 ug/day). The amounts of essential trace elements were very low in the infusions, and the estimated daily intake was calculated as 0.06 mg/day, 0.07 mg/day, 3.36 μg/day, and 23 μg/day for Fe, Zn, Cr, and Cu, respectively. It is clearly calculated that there is no health risk associated with the consumption of these herbal products with respect to their metal contents. However, the content of Pb and Cd must be monitored frequently to avoid the accumulation of these toxic elements.Because of the toxic properties of lead and cadmium, it is important to evaluate the safety of the herbal preparation. In this work the two toxic elements lead and cadmium were quantified in the raw materials of herbal remedies with average values close to or slightly above their permissible limits of 10 mg/kg and 0.3 mg/kg, respectively. It was found that 40% of the analyzed samples contain more than 10 mg/kg of lead and 60% of the analyzed samples contain more than 0.3 mg/kg of cadmium, while 30% of the samples contain the two elements above their permissible limits. Indeed, the determination of total concentration of the toxic elements in the herbal raw materials gives indication of the toxicity of the herbal preparation, but this step is not sufficient in assessing the safety of these products because the consumers take these remedies as recommended by the producers as their infusion in hot water. In fact, all infusions were found to contain fewer amounts of metals than the raw materials, and so it is important to evaluate the infusions for their safety. By assuming the consumption of 200 ml infusion prepared from 2-scahets (2 g) two times a day, as recommended by the manufacturer, and by using the mean value of the % extraction efficiency for Pb and Cd, the estimated daily intake was calculated as 8.32 Quantification of nine metal elements  in the raw materials and water infusions of selected herbal remedies and comparison with the values in the literature revealed that the heavy metal contents of these commercially available products vary and are affected by the plant species. In the raw materials, Fe had the highest mean content (440 mg/kg) and Cd had the lowest mean content (0.5 mg/kg). Mn and Cu demonstrated relatively high extraction efficiencies to the infusions while Fe had the lowest extraction efficiency. Some raw herbal materials had lead and cadmium contents above the permissible limits. However, the water infusions of these medicinal preparations contain lower levels of lead and cadmium and consumption of the tested beverages poses no risk to human health.


Separate microcosms containing tet(M)-possessing bacteria with ciliates or HNFs were prepared. The copy number of ex-tetM in seawater in the ciliate microcosm rapidly increased until 3 d after the incubation, whereas that in the HNF microcosm showed a slower increase until 20 d. The copy number of ex-tetM was stable in both cases throughout the incubation period, suggesting that extracellular ARGs are preserved in the environment, even in the presence of grazers. Additionally, ARGs in bacterial cells were constant in the presence of grazers. These results suggest that ARGs are not rapidly extinguished in a marine environment under grazing stress.Extracellular DNA (exDNA) is released from bacterial cells through various processes. The antibiotic resistance genes (ARGs) coded on exDNA may be horizontally transferred among bacterial communities by natural transformation. We quantitated the released/leaked tetracycline resistance gene,  Antibiotic resistance is a serious and growing public health issue worldwide . Antibioet al.  is a marine bacterium of unknown abundance that may cause infections in fish and humans .The bacterium strain land Sea . This baland Sea , 36. StrParanophrys marina  and Cafeteria roenbergensis (IOW23) were isolated by Weber et al.  of a size appropriate to their buccal apparatus . Its cells are typically spherical and range between 4 and 8 μm in diameter . This species resembles C. minuta, C. marsupialis, and Aeronema sippewissettensis  and autoclaved, and supplemented with heat-killed ®), which contained 250 mL of filtered-autoclaved seawater collected from coastal seawater off Matsuyama, Ehime Prefecture, Japan  in July 2015. Seawater parameters were as follows: water temperature 20.7°C, pH 8.7, and salinity 30 (psu). An overnight bacterial culture was harvested by centrifugation, and cells were then washed twice and resuspended in filtered-autoclaved seawater. Bacterial cells  and protist cells (103–104 cells mL−1) were inoculated into microcosms. The initial community composition on d 0 in each microcosm is shown in P. marina, IOW15, and 04Ya311 was prepared. A microcosm without 04Ya311 was also prepared as a control. All microcosms were incubated at 20°C for 20 d in the dark.The terms co-culture and mono-culture are defined as a microcosm with more than two species and that with one species, respectively. Microcosm experiments were performed in duplicate in 500-mL narrow-mouth polycarbonate square bottles  after an incubation at 25°C overnight. SMX was used in this experiment to inhibit the growth of ciliate-associated bacteria. Ciliates and HNFs were directly counted according to a previous study (−1) and fluorescein iso-thiocyanate  for 10 min in the dark. Cells were collected on 0.8-μm black nucleopore membranes (Whatman), washed several times with ice-cold 0.5 M sodium carbonate buffer (pH 9.5), and then counted using an Olympus BX51 fluorescence microscope .The colony-forming unit (CFU) of 04Ya311 was counted on an MHB plate containing 2% NaCl and 60 μg mLus study . In brieet al. . A 0.6 volume of cold isopropanol was then added, followed by an incubation for 1 h on ice and then centrifugation at 10,000×g at 4°C for 15 min. The pellet was resuspended in TE buffer, an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1 [v/v/v]) was added, and the suspension was then centrifuged at 10,000×g for 5 min. Each supernatant was mixed with an equal volume of chloroform-isoamyl alcohol (24:1 [v/v]) and centrifuged again. DNA in the supernatant was then precipitated with sodium acetate and cold ethanol  at −20°C for 1 h, and centrifuged at 10,000×g for 15 min. The pellet was then washed twice with 70% ethanol, dried, and resuspended in TE buffer.Each subsample (15 mL) was filtered through 47-mm polycarbonate membranes with a pore size of 0.22 μm  to collect bacteria and protists cells. All cells on filters were processed as described by Dempster et al.  to extraet al. , and restet(M), was targeted to measure the copy number of extracellular and intracellular pAQU1. In the case of extracellular tet(M) (ex-tetM), the positive sign of tet(M) may not be derived from the entire plasmid pAQU1 because the plasmid carrying tet(M) may be degraded into fragments. Large extracellular plasmids are reported to be fragile and readily sheared into small pieces or digested by nucleases (tet(M) was normalized using hlyA, which is positive in 04Ya311, but not in protist-associated bacteria. Primer sets have previously been reported by Tamminen et al. (tet(M) , and by Rivas et al. . Quantitative real-time PCR (qPCR) was performed using the CFX96 Real-time System . The qPCR program for the tet(M) gene was described previously .The pAQU1 plasmid codes seven ARGs , among wucleases , 19. Thun et al.  for tet and the significance of differences was tested by a one-tailed Student’s t-test to compare pairs of time points using Excel software. Each qPCR value was the average of triplicate measurements with standard errors.Experiments for each protist species were performed in duplicate. Results are indicated as the average of two independent experiments. In statistical analyses, data from two species of ciliates or HNFs were combined (as 7–1.4×108 CFU mL−1. The number of ciliates was 2.1–5.1×103 cells mL−1 and HNFs was 6.6×103–2.7×104 cells mL−1. Cell densities in the mono-culture microcosms of bacteria (p<0.05) (4 (P. marina) and 8.7–9.9×104 cells mL−1 (P. virginianum) by d 3 (p<0.05) (8 to 6.5×107 CFU mL−1 by d 3 (5 cells mL−1 (p<0.05) on d 3 (p=0.06)  , while t(p<0.05) , and the1 by d 3 . However) on d 3 . Slight (p=0.06) , and rea1 CFU  and 8.0×103 (IOW15) to 4.4–5.0×104 and 2.6–3.1×104 cells mL−1, respectively, by d 3 . It then slightly decreased to the initial level (2 copies mL−1) to d 20 (1.48–3.46×103 copies mL−1) (p<0.05) in the HNF co-cultures  copy number normalized with hlyA (tet(M) copy number in the bacterial population was nearly constant with minor fluctuations, varying from 6.05×10−4 to 2.20×10−3 (−4) were observed in the microcosms of co-cultured 04Ya311 and P. marina, while microcosms with P. virginianum slightly decreased from 6.25×10−4 (d 0) to 3.59×10−5 (d 20) , which was calculated from data reported by Bien et al.  copies normalized by hlyA was calculated to be approximately 10−3 (The abundance of pAQU1 in the 04Ya311 population with ciliates and HNFs was assessed in cellular fractions by measuring the ith hlyA . In cult.20×10−3 . Minor f5 (d 20) . Minor fin IOW23 . The copely 10−3 2 to 4.58×104 gene copies L−1  increased to 0.24 μg mL−1 by d 12 with elevations in ciliates from 101 to more than 104 cells mL−1  in bacterial cells remained constant, although a slight decrease in tet(M) was observed in bacteria with P. virginianum  may form the high background of the environmental ARG pool. These results provide a better understanding of the formation of an ARG pool and the dissemination of ARGs in natural seawater.In conclusion, we herein identified novel aspects to show that ARGs released by grazing pressure may be preserved in seawater. ARGs on plasmids may continue to be possessed by bacterial cells in the presence of grazers. ex-tet(M) and bacterial


R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC from Methanobrevibacter millerae SM9 was cloned and expressed in Escherichia coli and biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen for α-ketoglutarate. Optimal activity was observed at pH 6.5. The apparent KM for coenzyme (NADH) was 55.1 μM, and for sulfopyruvate, it was 196 μM (for sulfopyruvate the Vmax was 93.9 μmol min−1 mg−1 and kcat was 62.8 s−1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.( Methanobrevibacter ruminantium, the uptake of CoM is an energy-dependent reaction [Coenzyme M is an essential cofactor for the final reaction in the methanogenesis pathway and in the production of methane catalyzed by methyl-coenzyme M reductase (MCR) . It is treaction , 5.l-sulfopyruvate, the product of the third step of the canonical pathway catalyzed by ComC [R)-phosphosulfolactate, which is then dephosphorylated by ComB to produce (R)-sulfolactate. ComC oxidizes (R)-sulfolactate to sulfopyruvate, which is then decarboxylated by ComDE to produce sulfoacetaldehyde. ComC is related by sequence to lactate/malate dehydrogenases, N-methyl-l-amino acid dehydrogenases, 2,3-diketo-l-gulonate reductases, ureidoglycolate dehydrogenases, and an uncharacterized clade of thermophilic archaeal proteins [S)-hydroxyglutaric acid, which is a component of methanopterin [Recently, it has been found that the biosynthesis of coenzyme M occurs through two pathways that differ in the steps leading to the production of  by ComC . In the  by ComC , 6. Thes by ComC , sulfola by ComC , and sul by ComC . The subproteins , 10–12. nopterin .Methanocaldococcus jannaschii genome sequence became available, two malate dehydrogenase genes (MJ1425 and MJ0490) were annotated, and the two enzymes were expressed and characterized by Graupner et al. [S)-malate) [Vmax and Vmax/KM for oxidation of (R)-sulfolactate by NAD+ to sulfopyruvate) and was classified as a sulfolactate dehydrogenase (EC 1.1.1.337) [M. jannaschii ComC (MJ1425) has been shown to also catalyze the NAD-dependent oxidation of 2-hydroxy-4-mercaptobutyric acid to 4-mercapto-2-oxobutyric acid, a precursor of the natural product 3-mercaptopropionic acid [M. jannaschii ComC (MJ1425) that should act as true sulfolactate dehydrogenases. One, assigned as a malate dehydrogenase from Methanothermus fervidus, has also been characterized  has been characterized and corresponds to MTH1205 [l-phosphoserine to l-cysteate (related to threonine synthase) [When the r et al. . Althoug-malate) , MJ1425 1.1.337) , 11. Mornic acid  identifi or MDH; ). The ot MTH1205 . In the ynthase) , 17. An ynthase) .Methanocaldococcus jannaschii (MJ1425) has revealed that the NADH binding pocket is not a typical Rossmann-fold type as found in the malate/lactate dehydrogenase family and represents a new class of NADH-based dehydrogenase [S) hydrogen [S)-isomers of substrates are indicated to be converted in the reduction reaction [Structural analysis of the ComC from the hyperthermophilic methanogen rogenase . In addirogenase . In the hydrogen . Only propanesulfonic acid (Mops), 1,3-bis(tris(hydroxymethyl)methylamino)propane or Bis-Tris propane (BTP), tris(2-carboxyethyl)phosphine (TCEP), and dithiothreitol (DTT) were purchased from Sigma-Aldrich (USA). Other common chemicals were obtained from ThermoFisher Scientific (NZ).Materials including sulfopyruvate, oxaloacetate, Methanobrevibacter millerae SM9 [μL; Stratagene, USA) in a 50 μL reaction with 0.2 μM of each primer, 0.3 μM dNTP, approximately 20 ng M. millerae SM9 DNA, and 1 × buffer. The PCR cycling parameters had an initial denaturation of 95°C for 2 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s, and 68°C for 40 s. The PCR product was purified using agarose DNA electrophoresis and a Wizard SV Gel and PCR kit . It was then inserted into pET151D using TOPO cloning in chemically competent Escherichia coli strain TOP 10F according to the manufacturer's instructions . Colonies were screened by colony PCR using pET151D T7 forward primer and the ComC reverse primer using 2.5 U Taq polymerase , and then the recombinant plasmids were isolated using alkaline lysis and purification with a Wizard SV Gel and PCR Clean-up kit . The plasmid used for expression was sequenced to verify that the gene was in frame and that the sequence was identical to the reference sequence and then transformed into E. coli Rosetta 2 cells .The ComC gene of erae SM9  was amplE. coli and purify the protein using nickel affinity chromatography, except for the following conditions. The lysis buffer was slightly different  Triton X-100, 20% (v/v) glycerol, 2 mM CaCl2, and 2 mM MgCl2), and lysis was performed on ice. Cell debris was removed by centrifugation , and the supernatant was filtered (0.8 and 0.22 μm). Buffer was exchanged to 20 mM Mops pH 7.0 containing 2 mM TCEP. Glycerol (10% v/v) was added to the purified protein; it was snap frozen in liquid nitrogen and stored at −85°C until further use.We followed the methods of Schofield et al.  to expreε 3070 M−1 cm−1) during the reduction of sulfopyruvate by ComC was monitored. Activity was measured at 37°C. M. millerae SM9 was isolated from the rumen of a sheep and has an optimal growth temperature of 38°C [μmol of NADH to NAD+ per minute under standard assay conditions.Spectrophotometric measurements and calculation of initial velocity were performed using a Cary 100 UV-vis spectrophotometer  with a thermostatted cuvette holder, using 1 cm path length quartz cuvettes. The consumption of NADH . Kinetics for NADH required variable concentration of the coenzyme (10–500 μM) and 500 μM of sulfopyruvate substrate. Sulfopyruvate kinetics with variable concentration (50–600 μM) were carried out using 400 μM NADH. A concentration of 0.08–0.25 μM ComC in the assay was chosen for accuracy and so as to obtain absorbance changes of about 0.1 to 0.2 per minute. The total volume of all assays was 200 μL. Assays were carried out in triplicate. Kinetic parameters were determined by fitting the data to the Michaelis-Menten equation using GraFit [Standard assay conditions are 0.08–0.25 g GraFit .μl) was at a concentration of 1 mg·ml−1, and the elution buffer was 50 mM Mops pH 7.0 containing 2 mM TCEP and 0.5 M KCl.We followed the methods of Schofield et al.  to deterWe followed the general methods of Schofield et al.  for elecM. millerae SM9 ComC was expressed in E. coli, purified using nickel affinity chromatography, and characterized. The apparent molecular mass of ComC was 75 kDa, determined by gel filtration chromatography. As the predicted molecular mass of His-tagged ComC is 40104 Da (376 amino acids), this indicates that ComC is dimeric in solution. Both the M. jannaschii ComC (MJ1425) [M. fervidus enzyme (Mfer0389) [In this study, the (MJ1425)  and the fer0389)  were alsM. jannaschii ComC in the reduction reaction was 8.0 [M. fervidus enzyme was assayed at pH 7.4 or 8.0 [M. marburgensis enzyme (MTBMAc15830) was assayed at pH 7.6 [Chromobacter salexigens ComC was characterized at pH 9.0 [The effect of pH on the activity of sulfopyruvate reduction by ComC, in the presence of 400 mM KCl, was investigated. Two different buffers (pH 4.0–6.5 citrate buffer and pH 6.5–9.5 BTP buffer) were used. Optimal activity was observed at pH 6.5 . For com was 8.0 , the M. 4 or 8.0 , 15, thet pH 7.6  and the t pH 9.0 . No pH oM. jannaschii ComC and the M. fervidus enzyme were characterized in 100 mM potassium phosphate [+ and phosphate concentrations, corresponding to the intracellular medium of M. fervidus cells, enhanced the thermostability of the M. fervidus enzyme [M. millerae SM9 is unknown, but it is not likely to significantly exceed the ionic strength typical for rumen fluid, which is similar to that of blood (0.15 M) [The activity of sulfopyruvate reduction by ComC at pH 6.5 was affected by the KCl concentration with the enzyme showing low activity in the absence of added KCl. The specific activity increased approximately 7-fold with the increasing KCl concentration in the assay and reached a maximum at 400 mM KCl, after which a progressive decrease in activity was observed . No datahosphate , 15. Hig(0.15 M) .α-ketoglutarate were tested as ComC substrates using the reduction reaction in the presence of NADH. Sulfopyruvate was the preferred substrate, so was used in the standard ComC assay. Oxaloacetate showed 31% of the specific activity seen for sulfopyruvate, and α-ketoglutarate showed approximately 0.2%. A similar substrate preference was seen for the M. jannaschii ComC which showed a 3-fold lower KM for sulfopyruvate compared to oxaloacetate  and a 30-fold higher Vmax/KM [α-ketoglutarate, the M. jannaschii ComC had a 50-fold lower KM  and a 400-fold higher Vmax/KM [M. fervidus enzyme had a slightly lower KM for sulfopyruvate compared to oxaloacetate and 7-fold higher Vmax/KM [Sulfopyruvate, oxaloacetate, and  Vmax/KM . While f Vmax/KM . Further Vmax/KM .M. millerae SM9 ComC showed less than 1% specific activity in the reduction reaction with NADPH compared to NADH. This bias for NADH was also found for the M. marburgensis enzyme which had negligible specific activity using NADPH compared to NADH in the presence of oxaloacetate (<0.4%) [M. jannaschii ComC which showed a 5-fold lower KM for sulfopyruvate in the presence of NADH compared to NADPH  and a 60-fold higher Vmax/KM [M. fervidus enzyme had a 3-fold lower KM for sulfopyruvate in the presence of NADH compared to NADPH [KM for NADH compared to NADPH in the presence of oxaloacetate [ (<0.4%) . NADH wa Vmax/KM . Additioto NADPH  and an aoacetate .M. millerae SM9 ComC (KM 196 μM) and NADH (KM 55.1 μM) with the sulfopyruvate value being 5-fold that found for M. jannaschii ComC (40 μM) [M. millerae SM9 ComC KM for sulfopyruvate is also higher than that for the M. fervidus enzyme (70 μM) [Vmax of the M. millerae SM9 ComC for sulfopyruvate was 93.9 U·mg−1, and for NADH, it was 73.1 U·mg−1. The Vmax value is lower than that for sulfopyruvate of the M. jannaschii ComC (370 U·mg−1) [M. fervidus enzyme (120 U·mg−1) [M. jannaschii ComC and the M. fervidus enzymes show substrate inhibition at very low sulfopyruvate concentrations  [M. millerae SM9 ComC, with possible substrate inhibition at 600 μM, that is, 3-fold the sulfopyruvate KM value.The kinetic parameters of the SM9 ComC  were obtSM9 ComC . The rat (40 μM) . The M.  (70 μM) . The Vma U·mg−1)  and simi U·mg−1) . The M. , resp.) . The resMethanobrevibacter is indicated to be the dominant genus of methanogens in the rumen of sheep and cows [Methanobrevibacter ruminantium strain DSM 1093 has revealed, interestingly, that this rumen methanogen does not contain an intact coenzyme M biosynthesis pathway [M. ruminantium is likely to use coenzyme M derived from other methanogens within the rumen. Lovley et al. [and cows , 20. Coe pathway . It cont pathway . Thus, My et al.  have desM. millerae SM9 ComC was assayed in the reverse reaction of coenzyme M biosynthesis using sulfopyruvate. The only other ComC to have been extensively characterized is from the thermophilic methanogen M. jannaschii [M. millerae SM9 ComC represents a potential means of screening compounds for their ability to inhibit methanogens and in so doing, help mitigate methane emissions from ruminants.We have reported here the cloning, expression, and biochemical characterization of ComC from a representative rumen methanogen. The activity of nnaschii , 11. Due