Effects of lipopolysaccharide dosing on bacterial community composition and fermentation in a dual-flow continuous culture system.

The objectives of this study were to evaluate the effects of lipopolysaccharide (LPS) dosing on bacterial fermentation and bacterial community composition (BCC), to set up a subacute ruminal acidosis (SARA) nutritional model in vitro, and to determine the best sampling time for LPS dosing in a dual-flow continuous culture system. Diets were randomly assigned to 6 fermentors in a replicated 3 × 3 Latin square with three 11-d experimental periods that consisted of 7 d for diet adaptation and 4 d for sample collection. Treatments were control diet (CON), wheat and barley diet (WBD) to induce SARA, and control diet + LPS (LPSD). Fermenters were fed 72 g of dry matter/d. The forage:concentrate ratio of CON was 65:35. The WBD diet was achieved by replacing 40% of dry matter of the CON diet with 50% ground wheat and 50% ground barley. The LPS concentration in LPSD was 200,000 endotoxin units, which was similar to that observed in cows with SARA. The SARA inducing and LPS dosing started at d 8. The BCC was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform (Illumina Inc., San Diego, CA). The LPSD and CON maintained pH above 6 for the entire experimental period, and the WBD kept pH between 5.2 and 5.6 for 4 h/d, successfully inducing SARA. Digestibility of neutral detergent fiber and crude protein in LPSD were not different from WBD but tended to be lower than CON. Lipopolysaccharide dosing had no effect on pool of VFA concentrations and profiles but decreased bacterial N; the pattern changes of VFA and LPS in LPSD started to increase and be similar to WBD 6 h after LPS dosing. Pool of LPS concentration was around 11-fold higher in WBD and 4-fold higher in LPSD than CON. In the solid fraction, the BCC of LPSD was different from WBD and tended to be different from CON. In the liquid fraction, the BCC was different among treatments. The LPS dosing increased the relative abundance of Succinimonas, Anaeroplasma, Succinivibrio, Succiniclasticum, and Ruminobacter, which are main gram-negative bacteria related to starch digestion. Our results suggest that LPS dosing does not affect pH alone. However, LPS could drive the development of SARA by affecting bacteria and bacterial fermentation. For future studies, samples are suggested to be taken 6 h after LPS dosing in a dual-flow continuous culture system.

Effects of replacing canola meal with solvent-extracted camelina meal on microbial fermentation in a dual-flow continuous culture system.

Camelina is an oil seed crop that belongs to the Brassica family (Cruciferae). Camelina meal is a by-product from the biofuel industry that contains on average 38% crude protein and between 10 to 20% of residual fat, which limits the inclusion levels of camelina meal in dairy cow diets as the main protein supplement. Thus, we conducted a solvent extraction on ground camelina seed on a laboratory scale. The objectives of this study were (1) to assess the effects of replacing canola meal (CM) with solvent-extracted camelina meal (SCAM) in lactating dairy cow diets; and (2) to determine the effects of SCAM on microbial fermentation and AA flow in a dual-flow continuous culture system. Diets were randomly assigned to 6 fermentors in a replicated 3 × 3 Latin square with three 10-d experimental periods consisting of 7 d for diet adaptation and 3 d for sample collection. Treatments were 0, 50, and 100% SCAM inclusion, replacing CM as the protein supplement. Diets contained 55:45 forage:concentrate, and fermentors were fed 72 g of dry matter/d equally divided in 2 feeding times. On d 8, 9, and 10 of each period, samples were collected for analyses of pH, volatile fatty acids (VFA), N metabolism, NH3-N, digestibility, and AA flow. Statistical analysis was performed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC), and linear and quadratic effects of SCAM inclusion were assessed. Total VFA concentration and pH were not affected by diets. Molar proportion of acetate decreased, whereas molar proportion of propionate increased with SCAM inclusion. Total branched-chain VFA concentration was the least in fermentors fed diet 0, and greatest in fermentors fed diet 50. Digestibility of NDF decreased in fermentors fed SCAM diets, and dry matter, organic matter, and crude protein true digestibility were similar across diets. Concentration of NH3-N linearly decreased, and non-NH3-N linearly increased with SCAM inclusion. Bacterial efficiency (calculated as g of bacterial N flow/kg of organic matter truly digested) tended to be greater in fermentors fed diet 100. Outflow of Arg linearly increased with SCAM inclusion, whereas overall AA flow was not affected by diet. In conclusion, replacing CM with SCAM increased propionate molar proportion and non-NH3-N flow, and decreased NH3-N flow and concentration, which may improve animal energy status and N utilization. Inclusion of SCAM did not change most AA flow, indicating that it can be a potential replacement for CM.

Effect of replacing calcium salts of palm oil with camelina seed at 2 dietary ether extract levels on digestion, ruminal fermentation, and nutrient flow in a dual-flow continuous culture system.

Camelina is a drought- and salt-tolerant oil seed, which in total ether extract (EE) contains up to 74% polyunsaturated fatty acids. The objective of this study was to assess the effects of replacing calcium salts of palm oil (Megalac, Church & Dwight Co. Inc., Princeton, NJ) with camelina seed (CS) on ruminal fermentation, digestion, and flows of fatty acids (FA) and AA in a dual-flow continuous culture system when supplemented at 5 or 8% dietary EE. Diets were randomly assigned to 8 fermentors in a 2 × 2 factorial arrangement of treatments in a replicated 4 × 4 Latin square design, with four 10-d experimental periods consisting of 7 d for diet adaptation and 3 d for sample collection. Treatments were (1) calcium salts of palm oil supplementation at 5% EE (MEG5); (2) calcium salts of palm oil supplementation at 8% EE (MEG8); (3) 7.7% CS supplementation at 5% EE (CS5); and (4) 17.7% CS supplementation at 8% EE (CS8). Diets contained 55% orchardgrass hay, and fermentors were fed 72 g of dry matter/d. On d 8, 9, and 10 of each period, digesta effluent samples were taken for ruminal NH3, volatile fatty acids, nitrogen metabolism analysis, and long-chain FA and AA flows. Statistical analysis was performed using the MIXED procedure (SAS Institute Inc., Cary, NC). We detected an interaction between FA source and dietary EE level for acetate, where MEG8 had the greatest molar proportion of acetate. Molar proportions of propionate were greater and total volatile fatty acids were lower on CS diets. Supplementation of CS decreased overall ruminal nutrient true digestibility, but dietary EE level did not affect it. Diets containing CS had greater biohydrogenation of 18:2 and 18:3; however, biohydrogenation of 18:1 was greater in MEG diets. Additionally, CS diets had greater ruminal concentrations of trans-10/11 18:1 and cis-9,trans-11 conjugated linoleic acid. Dietary EE level at 8% negatively affected flows of NH3-N (g/d), nonammonia N, and bacterial N as well as the overall AA outflow. However, treatments had minor effects on individual ruminal AA digestibility. The shift from acetate to propionate observed on diets containing CS may be advantageous from an energetic standpoint. Moreover, CS diets had greater ruminal outflow of trans-10/11 18:1 and cis-9,trans-11 conjugated linoleic acid than MEG diets, suggesting a better FA profile available for postruminal absorption. However, dietary EE at 8% was deleterious to overall N metabolism and AA outflow, indicating that CS can be fed at 5% EE without compromising N metabolism.

Function of proline residues of MotA in torque generation by the flagellar motor of Escherichia coli.

Bacterial flagellar motors obtain energy for rotation from the membrane gradient of protons or, in some species, sodium ions. The molecular mechanism of flagellar rotation is not understood. MotA and MotB are integral membrane proteins that function in proton conduction and are believed to form the stator of the motor. Previous mutational studies identified two conserved proline residues in MotA (Pro 173 and Pro 222 in the protein from Escherichia coli) and a conserved aspartic acid residue in MotB (Asp 32) that are important for function. Asp 32 of MotB probably forms part of the proton path through the motor. To learn more about the roles of the conserved proline residues of MotA, we examined motor function in Pro 173 and Pro 222 mutants, making measurements of torque at high load, speed at low and intermediate loads, and solvent-isotope effects (D2O versus H2O). Proton conduction by wild-type and mutant MotA-MotB channels was also assayed, by a growth defect that occurs upon overexpression. Several different mutations of Pro 173 reduced the torque of the motor under high load, and a few prevented motor rotation but still allowed proton flow through the MotA-MotB channels. These and other properties of the mutants suggest that Pro 173 has a pivotal role in coupling proton flow to motor rotation and is positioned in the channel near Asp 32 of MotB. Replacements of Pro 222 abolished function in all assays and were strongly dominant. Certain Pro 222 mutant proteins prevented swimming almost completely when expressed at moderate levels in wild-type cells. This dominance might be caused by rotor-stator jamming, because it was weaker when FliG carried a mutation believed to increase rotor-stator clearance. We propose a mechanism for torque generation, in which specific functions are suggested for the proline residues of MotA and Asp32 of MotB.

The solubility of toluene in aqueous salt solutions.

The solubility of toluene has been measured in distilled water, and in various inorganic salt solutions. Values of the Setschenow constant, K(S), which quantify toluene solubility versus salt concentration, have been determined for each salt. Values of K(S) are compared to the activity of water for the salt solutions. Data from this study, consistent with earlier data, suggests that the effects of salts upon toluene solubility are non-additive. This contrasts the additive behavior of inorganic salts upon the solubility of nonpolar organic compounds, such as benzene and naphthalene, reported in the literature. Specific interaction between slightly polar toluene and ions in solution is suggested as a possible explanation for the non-additive effect of salts on the solubility of toluene.

Estimation of Koc values for deuterated benzene, toluene, and ethylbenzene, and application to ground water contamination studies.

Sorption partition coefficients between water and organic carbon (Koc) for deuterated benzene, toluene, and ethylbenzene have been estimated by measuring values of the octanol-water partition coefficient (Kow) and HPLC retention factors (k1), which correlate closely to values of Koc. Measured values of log Kow for non-deuterated and deuterated toluene are 2.77 (+/- 0.02) and 2.78 (+/- 0.04), respectively, indicating that within experimental error, log Koc for deuterated and non-deuterated toluene are the same. The HPLC method provides greater precision, and yields values of delta log Koc (= log Koc [deuterated]-log Koc [non-deuterated]) of -0.021 (+/- 0.001) for benzene, -0.028 (+/- 0.002) for toluene, and -0.035 (+/- 0.003) for ethylbenzene. The small values of delta log Koc demonstrates that deuterated compounds are excellent tracers for the hydrologic behavior of ground water contaminants.

Aqueous complexing of nickel and zinc with 3-(N-morpholino)propanesulfonic acid and the solubility products of nickel and zinc hydroxides.

Phosphate and carbonate buffers serve as excellent pH buffers at circumneutral values of pH, except for systems requiring significant concentrations of heavy metals, which are severely restricted by the low solubilities of heavy metal phosphates and carbonates. 3-(N-morpholino)propanesulfonic acid (MOPS; pK(a) 7.2) is a suitable replacement buffer at circumneutral values of pH. However, only limited data are available concerning aqueous complexing involving MOPS. Aqueous complexing of nickel and zinc with MOPS has been investigated by the solubility method, using the sparingly soluble solids Ni(OH)(2) and Zn(OH)(2). Log K(sp) values of -15.9 and -16.6 have been determined for Ni(OH)(2) and Zn(OH)(2) respectively. Experiments with MOPS indicate that there is negligible complexing between Zn and MOPS, but that significant Ni-MOPS complexing occurs, such that Ni(2+) + OH(-) + MOPSNi(OH)MOPS composite function: log K = +7.0.