Substrate Kinetics Research Articles

Cynomolgus Monkey as a Clinically Relevant Model to Study Transport Involving Renal Organic Cation Transporters: In Vitro and In Vivo Evaluation.

Organic cation transporter (OCT) 2, multidrug and toxin extrusion protein (MATE) 1, and MATE2K mediate the renal secretion of various cationic drugs and can serve as the loci of drug-drug interactions (DDI). To support the evaluation of cynomolgus monkey as a surrogate model for studying human organic cation transporters, monkey genes were cloned and shown to have a high degree of amino acid sequence identity versus their human counterparts (93.7, 94.7, and 95.4% for OCT2, MATE1, and MATE2K, respectively). Subsequently, the three transporters were individually stably expressed in human embryonic kidney (HEK) 293 cells and their properties (substrate selectivity, time course, pH dependence, and kinetics) were found to be comparable to the corresponding human form. For example, six known human cation transporter inhibitors, including pyrimethamine (PYR), showed generally similar IC50 values against the monkey transporters (within sixfold). Consistent with the in vitro inhibition of metformin (MFM) transport by PYR (IC50 for cynomolgus OCT2, MATE1, and MATE2K; 1.2 ± 0.38, 0.17 ± 0.04, and 0.25 ± 0.04 µM, respectively), intravenous pretreatment of monkeys with PYR (0.5 mg/kg) decreased the clearance (54 ± 9%) and increased in the area under the plasma concentration-time curve of MFM (AUC ratio versus control = 2.23; 90% confidence interval of 1.57 to 3.17). These findings suggest that the cynomolgus monkey may have some utility in support of in vitro-in vivo extrapolations (IVIVEs) involving the inhibition of renal OCT2 and MATEs. In turn, cynomolgus monkey-enabled IVIVEs may inform human DDI risk assessment.

Treatment of wastewater from water based paint industries using submerged attached growth reactor

Pollutants generated from water based paint industries contain mixture of toxic organic solvents. The present study dealt with individual and simultaneous degradation of ethylene glycol, propylene glycol and isobutanol from water based paint industry in batch and continuous mode. Biodegradation of toxic organic solvents under aerobic condition using acclimatized microbial consortia was evaluated in a batch system independently. Haldane biokinetic model stimulated the biodegradation kinetics of single substrate system satisfactorily. The biodegradation kinetics of mixed substrate was stimulated using the biokinetic parameters from single-substrate biodegradation studies. Competitive inhibition model performed well among the various models used for multi-substrate systems. Further models were statistically evaluated using the modified efficiency of coefficient (E). Performance of submerged attached bioreactor at different air flow and organic loading rates, on the removal efficiency was evaluated continuously. It is found that at an organic loading rate of 0.89 kg COD/m3/d, the maximum removal efficiency of 97% was achieved. Studies indicated that the removal efficiency was greatly depending on organic loading rate (OLR) and pollutant nature. Overall, experimental results demonstrated that the submerged attached growth reactor used in present study is found to be viable and efficient treatment system for the wastewater from water based paint industries.

Limits to Dihydrogen Incorporation into Electron Sinks Alternative to Methanogenesis in Ruminal Fermentation.

Research is being conducted with the objective of decreasing methane (CH4) production in the rumen, as methane emissions from ruminants are environmentally damaging and a loss of digestible energy to ruminants. Inhibiting ruminal methanogenesis generally results in accumulation of dihydrogen (H2), which is energetically inefficient and can inhibit fermentation. It would be nutritionally beneficial to incorporate accumulated H2 into propionate or butyrate production, or reductive acetogenesis. The objective of this analysis was to examine three possible physicochemical limitations to the incorporation of accumulated H2 into propionate and butyrate production, and reductive acetogenesis, in methanogenesis-inhibited ruminal batch and continuous cultures: (i) Thermodynamics; (ii) Enzyme kinetics; (iii) Substrate kinetics. Batch (N = 109) and continuous (N = 43) culture databases of experiments with at least 50% inhibition in CH4 production were used in this meta-analysis. Incorporation of accumulated H2 into propionate production and reductive acetogenesis seemed to be thermodynamically feasible but quite close to equilibrium, whereas this was less clear for butyrate. With regard to enzyme kinetics, it was speculated that hydrogenases of ruminal microorganisms may have evolved toward high-affinity and low maximal velocity to compete for traces of H2, rather than for high pressure accumulated H2. Responses so far obtained to the addition of propionate production intermediates do not allow distinguishing between thermodynamic and substrate kinetics control.

Optimization of continuous microbiological synthesis processes with nonlinear microbial growth kinetics

A methodology for solving the optimization problem for a widespread microbiological synthesis process is considered, and a numerical solution of the problem is presented. The methodology developed is based on substrate and product inhibition kinetics. The optimization problem is solved using the target component productivity Qp as the optimality criterion. Three variants of optimization problem formulation are considered: optimization of substrate concentration in the feed stream at a fixed dilution rate; dilution rate optimization at a fixed substrate concentration in the feed, Sf; optimization with respect to two variables—Sf and Qp. A specific feature of the solution is that it is necessary to check whether the optimal values obtained are usable in the practical implementation of the synthesis process. Numerical results of optimization are presented for the three variants of problem formulation.

Effects of substrate concentration on methane potential and degradation kinetics in batch anaerobic digestion

In this study, two experiments were conducted to evaluate the impact of substrate concentrations on methane potential and degradation kinetics of substrate. The biochemical methane potential (BMP) tests in Experiment I were performed at a constant inoculum to substrate ratio (ISR), whereas, different ISRs were applied in Experiment II. Results obtained from Experiment I revealed that methane potential of substrate increased at a saturating trend with higher substrate concentrations, and could differ by up to 30% between the lowest and highest investigated concentrations. The results of Experiment II verified the results of Experiment I, and further showed that this trend also occurs when the substrate concentration is regulated with ISRs. In contrast, substrate concentration had no significant impact on the degradation kinetics. It was concluded that dilutions should be avoided when the substrate concentration is lower than 10gVS/L in order to avoid underestimations of methane potential from BMP test.

High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation

Full kinetic models provide insight into enzyme mechanism and kinetics and also support bioconversion process design and feasibility assessment. Previously we have established automated microwell methods for rapid data collection and hybrid kinetic modelling techniques for quantification of kinetic constants. In this work these methods are applied to explore the substrate selectivity and kinetics of monoamine oxidase, MAO-N-D5, from Aspergillus niger. In particular we examine the MAO-N-D5 variant Ile246Met/Asn336Ser/Met348Lys/Thr384Asn to allow the oxidation of secondary amines Initial screening showed that MAO-N-D5 enabled the selective oxidation of secondary amines in 8 and 9 carbon rings, as well as primary ethyl and propyl amines attached to secondary amines of indolines and pyrrolidines. Subsequently we developed a first kinetic model for the MAO-N-D5 enzyme based on the ping-pong bi-bi mechanism (similar to that for the human MAO-A enzyme). The full set of kinetic parameters were then established for three MAO-N-D5 substrates namely; 3-azabicyclo[3,3,0]octane, 1-(2 amino ethyl) pyrrolidine and 3-(2,3-dihydro-1H-indole-1-yl)propan-1-amine. The models for each amine substrate showed excellent agreement with experimentally determined progress curves over a range of operating conditions. They indicated that in each case amine inhibition was the main determinant of overall reaction rate rather than oxygen or imine (product) inhibition. From the perspective of larger scale bioconversion process design, the models indicated the need for fed-batch addition of the amine substrate and to increase the dissolved oxygen levels in order to maximize bioconversion process productivity.

Mechanistic Insights of a Concerted Metalation–Deprotonation Reaction with [Cp*RhCl2]2

The effect of the carboxylate used in a concerted metalation–deprotonation reaction is probed and shows a direct correlation of pKa to observed rate up to a pKa of 4.3, where the rate drops off at higher pKa. The rate of the C–H activation of 2-(4-methoxyphenyl)pyridine with [Cp*RhCl2]2 and carboxylate follows first-order kinetics in the active metal species, Cp*RhCl(κ2-OAc), and zero-order kinetics in substrate when in a 1:1 ratio. There is a first-order dependence on substrate observed when excess substrate is present. The evaluation of the mechanism using kinetic studies allowed for a mechanistic proposal in which a second Ph′Py coordinates prior to the rate-determining C–H activation.

Evaluation of methane production features and kinetics of Bougainvillea spectabilis Willd waste under mesophilic conditions.

The objective of this study was to investigate and evaluate the biomethane potential (BMP) of Bougainvillea spectabilis Willd waste in Yunnan, China, when subjected to mesophilic anaerobic digestion (AD). Three different categories of plant waste investigated were as follows: the flowers, leaves and stems of B. spectabilia. These three portions were assessed for their BMP in a laboratory-scale batch anaerobic digester for a period of 60 days at 30±0.1°C temperature. The results show that maximum daily methane yield of B. spectabilia’s flowers, leaves and stems were 65.95, 56.29 and 18.8 mL/(g.VS), respectively. Moreover, the research used substrate kinetics, including the Gompertz equation, the logistic equation and the transference function to analyze the AD process. All models fit the experimental data with R2>0.993. However, the Gompertz equation presented the best agreement in the fitting progress. Key words: Bougainvillea spectabilis Willd, biomethane potential, anaerobic digestion, mathematical model.

Enzyme Module Systems for the Synthesis of Uridine 5′‐Diphospho‐α‐D‐glucuronic Acid and Non‐Sulfated Human Natural Killer Cell‐1 (HNK‐1) Epitope

AbstractTailor‐made strategies for the stereo‐ and regioselective multi‐step enzymatic synthesis of glycoconjugates require well characterized glycosyltransferases and carbohydrate modifying enzymes. We here report on a novel enzyme cascade for the synthesis of uridine 5′‐diphospho‐α‐D‐glucuronic acid (UDP‐GlcA) and the non‐sulfated human natural killer cell‐1 (HNK‐1) epitope including in situ regeneration of UDP‐GlcA and the cofactor nicotinamide adenine dinucleotide NAD+ by the combination of four enzymes in one‐pot. In the first enzyme module sucrose synthase 1 (SuSy1) is used to produce uridine 5′‐diphospho‐α‐D‐glucose (UDP‐Glc) from sucrose and uridine 5′‐diphosphate (UDP). The combination with UDP‐Glc dehydrogenase in the second enzyme module leads to the synthesis of UDP‐GlcA with concomitant in situ regeneration of the cofactor NAD+ by nicotinamide adenine dinucleotide hydride (NADH)‐oxidase. In the third enzyme module the mammalian glucuronyltransferase GlcAT‐P catalyzes the synthesis of the non‐sulfated HNK‐1 epitope by regioselective transfer of GlcA onto N‐acetyllactosamine type 2 (LacNAc type 2). We present a comprehensive study on substrate kinetics, substrate specificities, variation and relation of enzyme activities as well as cross inhibition of intermediate products. With optimized reaction conditions we obtain superior product yields with streamlined synthesis costs for the expensive nucleotide sugar UDP‐GlcA and cofactor NAD+.magnified image

Kinetics of Substrate Biodegradation under the Cumulative Effects of Bioavailability and Self-Inhibition.

Microbial degradation is an important process in many environments controlling for instance the cycling of nutrients or the biodegradation of contaminants. At high substrate concentrations toxic effects may inhibit the degradation process. Bioavailability limitations of a degradable substrate can therefore either improve the overall dynamics of degradation by softening the contaminant toxicity effects to microorganisms, or slow down the biodegradation by reducing the microbial access to the substrate. Many studies on biodegradation kinetics of a self-inhibitive substrate have mainly focused on physiological responses of the bacteria to substrate concentration levels without considering the substrate bioavailability limitations rising from different geophysical and geochemical dynamics at pore-scale. In this regard, the role of bioavailability effects on the kinetics of self-inhibiting substrates is poorly understood. In this study, we theoretically analyze this role and assess the interactions between self-inhibition and mass transfer-limitations using analytical/numerical solutions, and show the findings practical relevance for a simple model scenario. Although individually self-inhibition and mass-transfer limitations negatively impact biodegradation, their combined effect may enhance biodegradation rates above a concentration threshold. To our knowledge, this is the first theoretical study describing the cumulative effects of the two mechanisms together.

Fluorogenic sequencing using halogen-fluorescein-labeled nucleotides.

Fluorogenic sequencing is a sequencing-by-synthesis technology that combines the advantages of pyrosequencing and fluorescence detection. With native duplex DNA as the major product, we employ polymerase to incorporate the complement- arily matched terminal phosphate-labeled fluorogenic nucleotides into the DNA template and release halogen-fluorescein as the reporter. This red-emitting fluorophore successfully avoids spectral overlap with the autofluorescence background of the flow chip. We fully characterized the enzymatic reaction kinetics of the new substrates, and performed a 35-base sequencing experiment with 60 reaction cycles. Our achievement expands the substrate repertoire for fluorogenic sequencing, and extends the spectral range to obtain better signal-to-background performance.

Photocatalytic degradation of three amantadine antiviral drugs as well as their eco-toxicity evolution

Abstracts Advanced oxidation processes (AOPs) relying on in situ generated highly reactive OH are successfully applied to water purification. The absolute reaction rate constants for OH with three antiviral drugs were first reported through pulsed radiolysis experiments. Results found that OH reacted quickly with these substrates, with bimolecular reaction rate constants of 6.31 × 10 9 , 5.13 × 10 9 , and 7.05 × 10 9 M −1 s −1 for 1-amantadine, 2-amantadine, and rimantadine, respectively. The photocatalytic degradation kinetics of substrates were followed pseudo-first-order kinetics according to Langmuir–Hinshelwood model in TiO 2 suspensions, and the apparent rate constants were obtained as 0.076, 0.084, and 0.102 min −1 for three antiviral drugs, respectively. Scavenger experiments revealed that OH was the major reactive species involved in antiviral drugs degradation. To probe the photocatalytic degradation mechanism, the fate of nitrogen elements and the change of total organic carbon were also examined, and the data showed that all three drugs could be completely mineralized into CO 2 , H 2 O, and inorganic ions (NO 3 − and NH 4 + ) without generating any detectable products with enough degradation time. To further insight into the potential adverse effect of three antiviral drugs and their degradation products, the acute aquatic toxicity of degradation solutions were evaluated at three different trophic levels, and the toxicities first increased slightly and then decreased rapidly as the total organic carbon decreased.

Characterization of Anaerobic Degradability and Kinetics of Harvested Submerged Aquatic Weeds Used for Nutrient Phytoremediation

In this study, eight different submerged aquatic species were screened by batch biochemical methane potential and anaerobic degradability tests to identify a promising/suitable feedstock. Kinetics of the best-screened substrate were studied in a mesophilic semi-continuous experiment. The aquatic species Myriophyllum aquaticum, Egeria densa and Potamogeton perfoliatus showed relatively higher methane yields of over 400 NmL/g-VS (volatile solids). Semi-continuous operation was carried out by feeding E. densa for over 400 days. The achieved results were 33%–53% chemical oxygen demand (COD) reduction and methane yield of 126–231 NmL/g-VS with a short hydraulic retention time (HRT). Additionally, the NH4+ and PO43− releases from the biomass to water were found to be low (18%–27% and 2.5%–3.9%) throughout the experiment. Hydrolysis was the limiting step in the digestion of E. densa, regardless of changes in HRT (15–45 days). The acid-phase model indicated that the hydrolysis rate constant (kh) of E. densa was 0.058 one/day, which was one third lower the kh value of food waste, but quite similar to cow manure.

Effect of asparagine substitutions in the YXN loop of a class C β-lactamase of Acinetobacter baumannii on substrate and inhibitor kinetics.

Class C cephalosporinases are a growing threat, and inhibitors of these enzymes are currently unavailable. Studies exploring the YXN loop asparagine in the Escherichia coli AmpC, P99, and CMY-2 enzymes have suggested that interactions between C6' or C7' substituents on penicillins or cephalosporins and this Asn are important in determining substrate specificity and enzymatic stability. We sought to characterize the YXN loop asparagine in the clinically important ADC-7 class C β-lactamase of Acinetobacter baumannii. Mutagenesis at the N148 position in ADC-7 yields functional mutants (N152G, -S, -T, -Q, -A, and -C) that retain cephalosporinase activity. Using standard assays, we show that N148G, -S, and -T variants possess good catalytic activity toward cefoxitin and ceftaroline but that cefepime is a poor substrate. Because N152 variants of CMY-2, another class C β-lactamase, are more readily inhibited by tazobactam due to higher rates of inactivation, we also tested if the N148 substitutions in ADC-7 would affect inactivation by sulfone inhibitors, sulbactam and tazobactam, class A β-lactamase, and A. baumannii penicillin-binding protein (PBP) inhibitors with in vitro activity against ADC-7. The 50% inhibitory concentrations (IC50s) for tazobactam and sulbactam were improved, with 7-fold and 2-fold reductions, respectively, for the N148S variant. A homology model of the N148S ADC-7 enzyme in a Michaelis-Menten complex with tazobactam showed a loss of interaction between N148 and the sulfone moiety of the inhibitor. We postulate that this may result in more-rapid secondary ring opening of the inhibitor, as the unbound sulfone is an excellent leaving group, leading to more-rapid formation of the stable linearized inhibitor.

Massively parallel determination and modeling of endonuclease substrate specificity.

We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution.

Cysteine transport through excitatory amino acid transporter 3 (EAAT3).

Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.

A Theoretical and Experimental Investigation of Wastewater Treatment for a Polyethylene Terephthalate Production Unit

In this research, mathematical modelling of an anaerobic hybrid bioreactor for effluent treatment of a PolyEthylene Terephthalate (PET) unit in a petrochemical complex was performed. The developed model included a combination of a biofilm model for describing the substrate kinetics; a fluidized bed model for determination of species profiles along the reactor length and a bioreactor model for particle distribution inside the reactor. The reactions performed in the bioreactor included; i) the polymers hydrolysis; ii) fermentation of the resulting monomers; iii) the volatile fatty acids fermentation to Acetate and Hydrogen and iv) the Methane formation as the final product. The reactor was divided into n-segments each being fully mixed and the changes of granule densities at each one caused by granules growth were neglected. On the other hand, the experimental data obtained from an industrial unit in which, the pH was controlled by adding NaHCO3 and NaOH while; a mixture of Urea, di-ammonium phosphate and molasses were utilized as substrates. The total flow rate inside the 2,200 m 3 bioreactor was set 180 m 3 /h with 0.7 m/h upflow velocity. The theoretical data determined from the developed model for the volatile fatty acids’ (VFAs) production while the corresponding experimental data obtained from the industrial unit. Moreover, the comparison between the bioreactor effluent’s COD determined through both the theoretical and experimental studies were presented. The current model reproduced the experimental data with mean error of 14 % which considering the complexity of the undertaken system was rather satisfying.

Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants.

BackgroundA previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities.ResultsIn vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions.ConclusionsWe demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.

Experimental results and integrated modeling of bacterial growth on an insoluble hydrophobic substrate (phenanthrene).

Metabolism of a low-solubility substrate is limited by dissolution and availability and can hardly be determined. We developed a numerical model for simultaneously calculating dissolution kinetics of such substrates and their metabolism and microbial growth (Monod kinetics with decay) and tested it with three aerobic phenanthrene (PHE) degraders: Novosphingobium pentaromativorans US6-1, Sphingomonas sp. EPA505, and Sphingobium yanoikuyae B1. PHE was present as microcrystals, providing non-limiting conditions for growth. Total PHE and protein concentration were tracked over 6-12 days. The model was fitted to the test results for the rates of dissolution, metabolism, and growth. The strains showed similar efficiency, with vmax values of 12-18 g dw g(-1) d(-1), yields of 0.21 g g(-1), maximum growth rates of 2.5-3.8 d(-1), and decay rates of 0.04-0.05 d(-1). Sensitivity analysis with the model shows that (i) retention in crystals or NAPLs or by sequestration competes with biodegradation, (ii) bacterial growth conditions (dissolution flux and resulting chemical activity of substrate) are more relevant for the final state of the system than the initial biomass, and (iii) the desorption flux regulates the turnover in the presence of solid-state, sequestered (aged), or NAPL substrate sources.

A fully automated incubation system for the measurement of gas production and gas composition

A fully automated in vitro incubation system consisting of 32 incubation bottles (125ml) each fitted with a pressure sensor and a solenoid valve was developed to monitor total gas production and hydrogen (H2) and methane (CH4) composition. Gas volume is calculated from the pressure readings collected by a dedicated computer and when the threshold pressure of 9kPa is reached the fermentation gases are released into a gas chromatograph via a sampling loop. Substrates were incubated at 10mg/ml in 60ml of a medium with a buffer to rumen fluid ratio of 4:1. Typically, 20–25 measurements of gas volume and composition are obtained from each bottle over a 48h incubation period. More than 60% of the measurements are obtained during the first 12h of fermentation when rumen fermentation is most active. This allows the same model to be fitted to the data to describe the gas and CH4 production data and directly compare their kinetics for various substrates. The overall repeatability of the system has been tested over 2 years using a ‘standard ryegrass hay’ sample that was incubated as an internal control in each incubation run. Overall, repeatability of gas and CH4 production was comparable to other incubation systems with a CV of 7.2% and 12.5% for total gas and CH4 production. However, within the dataset an annual pattern was detected in the parameters of the standard hay. Differences in CH4 emissions from various substrates were largely explained by differences in short chain fatty acid production.In an initial series of test the system was used to evaluate the effect of incubation buffers or rumen fluid donor species on gas emissions and fermentation end products. While no differences were found for the different buffers used, our results indicate sheep rumen fluid leads to a slightly lower production of CH4 when compared to rumen fluid from cattle.The system presented here is the first rumen fluid batch culture system that automatically measures total gas, CH4 and H2 production and is currently used as a screening tool for substrates or additives that have a potential to lower CH4 emissions from ruminants.