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PubMed2002

Microbial biohydrogenation of oleic acid to trans isomers in vitro.

Ruminant products are significant sources of dietary trans fatty acids. Trans fatty acids, including various conjugated linoleic acid isomers, have been shown to act as metabolic modifiers of lipid metabolism. Trans fatty acids originate from biohydrogenation of dietary unsaturated fatty acids by gut microbes; however, the exact synthetic pathways are unclear. It was our goal to examine the biohydrogenation pathway for oleic acid, where oleic acid is hydrogenated directly to stearic acid. Our objective in this study was to trace the time course of appearance of 13C in labeled oleic acid to determine if trans monoenes are formed from the 13C-labeled oleic acid or if the 13C appears only in stearic acid as described in reviews of earlier work. Enrichments were calculated from the mass abundance of 13C in major fatty acid fragments and expressed as a percentage of total carbon isotopomers. Significant 13C enrichment was found in stearic acid, oleic acid, trans-6, trans-7, and in all trans C18:1 in positions 9-16. We concluded that the biohydrogenation of oleic acid by mixed ruminal microbes involves the formation of several positional isomers of trans monoenes rather than only direct biohydrogenation to form stearic acid as previously described.

Animals↗
PubMed2004

Biohydrogenation of dietary n-3 PUFA and stability of ingested vitamin E in the rumen, and their effects on microbial activity in sheep.

The present study investigated the susceptibility of dietary n-3 PUFA to ruminal biohydrogenation, the stability of ingested vitamin E in the rumen and the subsequent uptake of PUFA and vitamin E into plasma. Six cannulated sheep were assigned to six diets over five 33 d periods, in an incomplete 6x5 Latin square. The diets, based on dried grass, were formulated to supply 50 g fatty acids/kg DM using three lipid sources: Megalac (calcium soap of palm fatty acid distillate; Volac Ltd, Royston, Herts., UK), linseed (formaldehyde-treated; Trouw Nutrition, Northwich, Ches., UK) and linseed-fish oil (formaldehyde-treated linseed+fish oil). The diets were supplemented with 100 or 500 mg alpha-tocopheryl acetate/kg DM. Fat source or level of vitamin E in the diet did not alter microbial activity in the rumen. Biohydrogenation of linoleic acid (18 : 3n-6; 85-90 %), linolenic acid (18 : 3n-3; 88-93 %), docosahexaenoic acid (22 : 6n-3; 91 %) and EPA (20 : 5n-3; 92 %) was extensive. Feeding formaldehyde-treated linseed elevated concentrations of 18 : 3n-3 in plasma, whilst 22 : 6n-3 and 20 : 5n-3 were only increased by feeding the linseed-fish oil blend. Duodenal recovery of ingested vitamin E was high (range 0.79-0.92 mg/mg fed). High dietary vitamin E was associated with increased plasma alpha-tocopherol (2.57 v. 1.46 microg/ml for 500 and 100 mg alpha-tocopheryl acetate/kg DM respectively), although all concentrations were low. Plasma vitamin E levels, however, tended to decrease as the type and quantity of PUFA in the diet increased. The present study illustrates that nutritionally beneficial PUFA in both fish and linseed oils are highly susceptible to biohydrogenation in the rumen. Although alpha-tocopheryl acetate resisted degradation in the rumen, plasma vitamin E status remained deficient to borderline, suggesting either that uptake may have been impaired or metabolism post-absorption increased.

Animal Nutritional Physiological Phenomena↗
PubMed2004

Ruminal biohydrogenation in Holstein cows fed soybean fatty acids as amides or calcium salts.

Fatty amides of high oleate fats and calcium salts of palm oil were reported to resist biohydrogenation by ruminal microorganisms. This study was conducted to determine whether converting polyunsaturated fat sources to amides and calcium salts had equal ability to resist biohydrogenation. A total mixed ration consisting of forage and concentrate contained (dry basis): 1) 2.45% soybean oil (SBO), 2) 2.75% calcium salt of SBO, 3) 2.75% amide of SBO, or 4) 2.75% of a mixture of the calcium salt and amide (80:20, wt/wt) of SBO. The 4 diets were fed ad libitum to 4 multiparous lactating Holstein cows fitted with ruminal cannulas in a 4 x 4 Latin square with 21-d periods. Omasal samples were taken to measure postruminal fatty acid content and determine the extent of ruminal biohydrogenation. Adding SBO to the diets as either calcium salts or amides increased omasal flow of C18:2 (n-6) from 25 to 39 g/d. Omasal flow of C18:1 increased from 36 to 49 g/d when SBO was fed to cows as calcium salts, but increased to 86 g/d when SBO was fed as amides. Adding the soybean amide to the diet more than doubled the delivery of C18:1 (n-9) to the omasum of lactating cows, but it also increased trans fatty acid production in the rumen accompanied by milk fat depression. In this study, calcium salts and amide derivatives of fatty acids were both effective in enhancing omasal flow of unsaturataed fatty acids in lactating dairy cows. Amides were more effective than calcium salts for increasing the postruminal flow of oleic acid.

Amides↗
PubMed1991

Synthesis and biohydrogenation of fatty acids by ruminal microorganisms in vitro.

Ruminal degradation, synthesis, and biohydrogenation of fatty acids were examined in vitro. Diets were incubated with ruminal contents, and changes of fatty acids were measured. Two fat supplements, a calcium soap and an animal-vegetable blend, were included in diets at various levels. Addition to diets of acetate and isoacids (collective term for certain short-chain acids) also were tested for effects on fatty acid synthesis. Overall, 6.6 mg of fatty acids/g of fat-free diet were synthesized during 24-h incubation regardless of supplementations. Fatty acids synthesized in greatest amounts were odd-numbered or branched chains, whereas chains of 16 and 18 carbons changed little, and chains shorter than 14 carbons decreased. Degradation of [1-14C]palmitic acid was negligible, as determined by recovery of the label in CO2 (.03%) and acetate (1.09%) after 4-h incubation with rumen contents. Biohydrogenation of fatty acids averaged 47% in diets containing calcium soap and 71% with animal-vegetable blend. Synthesis and biohydrogenation were similar to those measured previously in vivo, showing that in vitro measurements reliably predicted metabolism of fatty acids in vivo.

Animal Feed↗
PubMed1995

Steady-state rates of linoleic acid biohydrogenation by ruminal bacteria in continuous culture.

Ruminal biohydrogenation of linoleic acid was determined in fermenters with a continuous culture of microorganisms. Rates of biohydrogenation and changes of fatty acids in culture were measured during steady-state concentration of linoleic acid that was achieved by continuous infusion of linoleic acid into the fermenters. A number of trans and cis isomers were identified using a GLC equipped with an infrared detector. The infusion of linoleic acid resulted in a substantial increase in the content of trans-C18:1 and a lesser increase in cis-C18:1. the major trans peak consisted of a mixture of n-9 and n-7 isomers. Biohydrogenation of infused linoleic acid averaged 77%. There was evidence of fatty acid loss, as determined by a decrease in the recovery of linoleic acid after 8 h of infusion. Addition of C18:2n-6 had no major effect on the VFA production by ruminal microorganisms. The results were similar to those measured in vivo, indicating that artificial fermenters were reliable predictors of fatty acid metabolism in vivo.

Animal Feed↗
PubMed1995

Lipolysis and biohydrogenation of soybean oil in the rumen in vitro: inhibition by antimicrobials.

This experiment attempted to lower rumen lipolytic activity, biohydrogenating activity, or both using antimicrobial compounds. In vitro incubations were carried out with rumen fluid, 80 mg of soybean oil, and .5 g of commercial concentrates as substrate. Unless stated otherwise, the final concentrations of the additives in the incubation was 20 ppm. Lipolysis and biohydrogenation were determined by separation of triacylglycerols and FFA by TLC; the fatty acid composition of each was determined by GLC before and after incubation and with or without additive. With some of the antibiotics, lipolysis was inhibited 10 to 20%, and the most potent inhibitors were ionophores and amoxicillin. Biohydrogenation (including C18:1) decreased only for lasalocid, but no additive could prevent hydrogenation of linolenic acid liberated from triacylglycerols. Some additives decreased hydrogenation of linoleic acid, but only slightly. Lipolytic activity decreased VFA production more than the other potent additives (amoxicillin, avoparcin, lasalocid sodium, monensin, and salinomycin sodium). This result could indicate a more specific toxic effect on lipolytic microbes. Finally, different antimicrobials influenced fermentation patterns differently (VFA proportions and CH4 production), but shifts were always in accordance with stoichiometric principles.

Animals↗
PubMed2026

Exploring biohydrogen producing potential of Arctic ice and water through metagenomics and dark fermentation kinetics.

Cryospheric ecosystems in the high Arctic harbor largely unexplored microbiomes with significant biotechnological potential. The present study evaluates the biohydrogen production capabilities of the indigenous microbiome of Ny-Ålesund, Svalbard, using glacial ice and surface water samples. Dark fermentation batch assays were performed at 4 °C and 20 °C with 2-bromoethanesulfonate (BES), a methanogenic inhibitor, to track the succession of metabolic and taxonomic diversity. Metagenomic and functional analyses revealed that under 20 °C and BES conditions, psychrotolerant microbial communities maximize biohydrogen production to 85% of the total biogas produced, with an acetate-dominant fermentation pathway, as inferred from volatile fatty acid (VFA) analysis. This evolves into a highly coordinated system utilizing a coupled Rnf-nitrogenase route alongside Formate Hydrogenlyase and [FeFe]-hydrogenase pathways. Kinetic modelling using the Modified Gompertz equation, along with Q10 temperature-sensitivity indices, demonstrated a very high latent catalytic potential in these cold-adapted microbiomes. This study indicates that Arctic microbiomes are highly elastic thermodynamically and could serve as highly efficient, manipulatable biocatalysts for the environmental recovery of bioenergy through engineered low-temperature systems.

Fermentation↗
PubMed2005

Utilization of keratin-containing biowaste to produce biohydrogen.

A two-stage fermentation system was constructed to test and demonstrate the feasibility of biohydrogen generation from keratin-rich biowaste. We isolated a novel aerobic Bacillus strain (Bacillus licheniformis KK1) that displays outstanding keratinolytic activity. The isolated strain was employed to convert keratin-containing biowaste into a fermentation product that is rich in amino acids and peptides. The process was optimized for the second fermentation step, in which the product of keratin fermentation--supplemented with essential minerals--was metabolized by Thermococcus litoralis, an anaerobic hyperthermophilic archaeon. T. litoralis grew on the keratin hydrolysate and produced hydrogen gas as a physiological fermentation byproduct. Hyperthermophilic cells utilized the keratin hydrolysate in a similar way as their standard nutrient, i.e., bacto-peptone. The generalization of the findings to protein-rich waste treatment and production of biohydrogen is discussed and possible means of further improvements are listed.

Aerobiosis↗
PubMed2006

Efficient induction of formate hydrogen lyase of aerobically grown Escherichia coli in a three-step biohydrogen production process.

A three-step biohydrogen production process characterized by efficient anaerobic induction of the formate hydrogen lyase (FHL) of aerobically grown Escherichia coli was established. Using E. coli strain SR13 (fhlA (++), DeltahycA) at a cell density of 8.2 g/l medium in this process, a specific hydrogen productivity (28.0 +/- 5.0 mmol h(-1) g(-1) dry cell) of one order of magnitude lower than we previously reported was realized after 8 h of anaerobic incubation. The reduced productivity was attributed partly to the inhibitory effects of accumulated metabolites on FHL induction. To avoid this inhibition, strain SR14 (SR13 DeltaldhA DeltafrdBC) was constructed and used to the effect that specific hydrogen productivity increased 1.3-fold to 37.4 +/- 6.9 mmol h(-1) g(-1). Furthermore, a maximum hydrogen production rate of 144.2 mmol h(-1) g(-1) was realized when a metabolite excretion system that achieved a dilution rate of 2.0 h(-1) was implemented. These results demonstrate that by avoiding anaerobic cultivation altogether, more economical harvesting of hydrogen-producing cells for use in our biohydrogen process was made possible.

Aerobiosis↗
PubMed2006

Differential biohydrogenation and isomerization of [U-(13)C]oleic and [1-(13)C]oleic acids by mixed ruminal microbes.

The additional mass associated with 13C in metabolic tracers may interfere with their metabolism. The comparative isomerization and biohydrogenation of oleic, [1-(13)C]oleic, and [U-13C]oleic acids by mixed ruminal microbes was used to evaluate this effect. The percent of stearic, cis-14 and -15, and trans-9 to -16 18:1 originating from oleic acid was decreased for [U-(13)C]oleic acid compared with [1-(13)C]oleic acid. Conversely, microbial utilization of [U-(13)C]oleic acid resulted in more of the 13C label in cis-9 18:1 compared with [1-(13)C]oleic acid (53.7 vs. 40.1%). The isomerization and biohydrogenation of oleic acid by ruminal microbes is affected by the mass of the labeled tracer.

Analysis of Variance↗
PubMed2006

Inhibition of biohydrogen production by ammonia.

Ammonia inhibition of biohydrogen production was investigated in batch and continuous flow reactors with glucose as a substrate. In batch tests, biohydrogen production rate was highly dependent on pH and ammonia (defined as the sum of NH3 of NH4+ species) concentrations above 2 g N/L. At pH = 6.2, the maximum production decreased from 56 mL/h at 2 g N/L to 16 mL/h at 10 g N/L. At pH = 5.2, production decreased from 49 mL/h (2g N/L) to 7 mL/h (16 g N/L). Hydrogen yield remained relatively constant in batch tests, varying from 0.96 to 1.17 mol-H2/mol-glucose. In continuous flow tests, both hydrogen production rates and yields were adversely affected by ammonia. When the reactor (2.0 L) was first acclimated under batch conditions to a low nitrogen concentration (<0.8 g N/L), H2 production and yields under continuous flow mode conditions were 170 mL/h and 1.9 mol-H2/mol-glucose, but decreased with increased ammonia concentrations up to 7.8 g N/L to 105 mL/h and 1.1 mol-H2/mol-glucose. There was no hydrogen production under continuous flow conditions if the reactor was initially operated under batch flow conditions at ammonia concentrations above 0.8 g N/L. It is concluded that the hydrogen production is possible at high concentrations (up to 7.8 g N/L) of ammonia in continuous flow systems as long as the reactor is initially acclimated to a lower ammonia concentration (<0.8 g N/L).

Ammonia↗
PubMed2005

Photosynthesis: a blueprint for solar energy capture and biohydrogen production technologies.

Solar energy capture, conversion into chemical energy and biopolymers by photoautotrophic organisms, is the basis for almost all life on Earth. A broad range of organisms have developed complex molecular machinery for the efficient conversion of sunlight to chemical energy over the past 3 billion years, which to the present day has not been matched by any man-made technologies. Chlorophyll photochemistry within photosystem II (PSII) drives the water-splitting reaction efficiently at room temperature, in contrast with the thermal dissociation reaction that requires a temperature of ca. 1550 K. The successful elucidation of the high-resolution structure of PSII, and in particular the structure of its Mn(4)Ca cluster provides an invaluable blueprint for designing solar powered biotechnologies for the future. This knowledge, combined with new molecular genetic tools, fully sequenced genomes, and an ever increasing knowledge base of physiological processes of oxygenic phototrophs has inspired scientists from many countries to develop new biotechnological strategies to produce renewable CO(2)-neutral energy from sunlight. This review focuses particularly on the potential of use of cyanobacteria and microalgae for biohydrogen production. Specifically this article reviews the predicted size of the global energy market and the constraints of global warming upon it, before detailing the complex set of biochemical pathways that underlie the photosynthetic process and how they could be modified for improved biohydrogen production.

Greenhouse Effect↗
PubMed1972

Quantitative aspects of fatty acid biohydrogenation, absorption and transfer into milk fat in the lactating goat, with special reference to the cis- and trans-isomers of octadecenoate and linoleate.

1. Surgically prepared lactating goats were used to obtain quantitative information on the biohydrogenation and absorption of dietary fat, and on the mammary uptake and transfer into milk fat of the complex mixture of cis- and trans-isomers of octadecenoate that arise during ruminal biohydrogenation. 2. About 90% of dietary linolenate, linoleate and oleate was hydrogenated in the rumen, and the availability to the animals of the essential fatty acid, linoleate, represented only 0.5-1.5% of the total dietary energy. 3. The intra-ruminal administration of (14)C-labelled linolenate and linoleate showed that these acids were not absorbed from the rumen, in agreement with previous work. 4. No selectivity was observed in the metabolism of the geometrical and positional isomers of octadecenoate: their rates of absorption from the small intestine, transfer into lymph, uptake by the mammary gland and appearance in milk fat were similar. 5. The desaturase activity of intestinal epithelium was demonstrated by the appearance in lymph of [1-(14)C]oleate after the addition of [1-(14)C]stearate to the small intestine.

Animals↗
PubMed2002

Characterization of 18:1 and 18:2 isomers produced during microbial biohydrogenation of unsaturated fatty acids from canola and soya bean oil in the rumen of lactating cows.

Ruminal production of biohydrogenation intermediates in response to unsaturated oils was assessed using 24 Jersey cows fed a control diet or the control diet supplemented at 35 g/kg dry matter (DM) with canola, soya bean, or a mixture of equal amounts of canola plus soya bean oil for 4-weeks. Total fatty acid content averaged 63 or 35 g/kg DM for oil-supplemented diets or control. Oleic acid accounted for 6, 29, 21 or 12 g/kg DM in the control, canola, mixture, or soya bean oil diet, respectively. Linoleic acid averaged 17, 19, 26, or 33 g/kg DM and linolenic acid 5, 5, 6 or 8 g/kg DM for control, canola, mixture, or soya bean oil. Concentrations of cis12-, trans11-, trans13+14, and trans15-18:1 were 0.81, 2.99, 2.24, and 0.73 mg/g rumen fluid, respectively, in response to soya bean oil and were 126, 90, 45, and 38% greater compared with other diets. Trans11cis15-, cis9trans11- and cis9 cis11-18:2 also were greater when soya bean oil (0.30, 0.34 and 0.01 mg/g, respectively) was fed compared with other treatments (0.12, 0.21 and 0.004 mg/g, respectively). Feeding canola oil resulted in greater concentrations of trans4-, trans5-, trans6+7+8-, trans9- and trans10-18:1 (0.20, 0.25, 0.87, 0.39 and 0.70 mg/g, respectively) compared with other diets (0.09, 0.15, 0.36, 0.20 and 0.46 mg/g, respectively). Trans10cis12-18:2 concentration did not differ as a result of diet and averaged 0.002 mg/g rumen contents. The pattern of 18:1 and 18:2 isomers formed during ruminal biohydrogenation depends greatly on dietary profile of unsaturated fatty acids.

Animal Feed↗
PubMed1992

Ruminal biohydrogenation of linoleoyl methionine and calcium linoleate in sheep.

Four ruminally and duodenally cannulated Hampshire wethers were used in a 4 x 4 Latin square experiment to determine whether linoleoyl methionine and calcium linoleate would increase duodenal flow of unsaturated fatty acids (C18:2 + cis C18:1). All animals received the same basal diet plus a treatment enclosed in gelatin capsules that were placed directly in the rumen. Of the four experimental treatments, one was a control (empty capsules) and three were 5 g of fatty acid equivalent as either free linoleic acid, calcium linoleate, or linoleoyl methionine. Linoleoyl methionine had the lowest ruminal disappearance of C18:2 + cis C18:1. Ruminal loss of unsaturated fatty acids from each supplement exclusive of feed unsaturated fatty acids was 69.8, 92.9, and 94.6% for linoleoyl methionine, free linoleic acid, and calcium linoleate, respectively. Duodenal flow of methionine also was higher for linoleoyl methionine than for control, free linoleic acid, or calcium linoleate (2.5, 1.7, 2.0, and 2.5 g/d, respectively). Plasma linoleic acid was higher for linoleoyl methionine than for control or free linoleic acid but was not different from calcium linoleate (22.0, 17.8, 18.9, and 20.2% of total fatty acids, respectively). Plasma methionine levels were not different among treatments. Intestinal disappearance of unsaturated fatty acids did not differ among treatments. Linoleoyl methionine resisted ruminal biohydrogenation and was digested normally in the intestine. Calcium linoleate did not escape biohydrogenation by ruminal bacteria.

Absorption↗
PubMed1995

Butylsoyamide protects soybean oil from ruminal biohydrogenation: effects of butylsoyamide on plasma fatty acids and nutrient digestion in sheep.

Based on previous results showing partial resistance of fatty acyl amides to ruminal biohydrogenation, butylsoyamide was added to sheep diets in an attempt to increase unsaturation of plasma fatty acids. Twelve wethers averaging 34 +/- 3.2 kg BW were randomly assigned to three diets containing either no added fat (control), 5% soybean oil, or 5% butylsoyamide. Dry matter intake was greater (P < .05) for sheep fed butylsoyamide than for sheep fed soybean oil (740 and 581 g/d, respectively), but neither fat supplement differed from the control diet (680 g/d). The soybean oil supplement reduced (P < .05) total VFA concentration (59.0 and 38.7 mM) and acetate:propionate (4.10 and 2.56) in ruminal samples compared with the control diet. Butylsoyamide had no effect (P > .05) on total VFA (54.4 mM) or acetate:propionate (2.96). Total tract ADF digestibility was not affected (P > .05) by either fat supplement. Relative to the control diet, soybean oil increased (P < .05) plasma linoleic acid concentration 22% compared with a 58% increase from feeding butylsoyamide (26.7, 32.6, and 42.1% of total fatty acids, respectively). Linoleic acid concentration in plasma neutral lipids, relative to the control diet, increased 15.8% (P < .05) for soybean oil compared with 64.9% (P < .05) for butylsoyamide (31.6, 36.6, and 52.1% of total fatty acids, respectively). Converting soybean oil triglycerides to fatty acyl amides substantially reduces negative effects of the oil on ruminal fermentation and increases unsaturated fatty acids in plasma. The increase in plasma unsaturated fatty acids demonstrates at least partial resistance of fatty acyl amides to ruminal biohydrogenation and their digestion and absorption postruminally.

Animal Feed↗
PubMed2005

Fatty acid profiles associated with microbial colonization of freshly ingested grass and rumen biohydrogenation.

Two in situ studies were conducted to examine the use of odd-chain fatty acid profiles to study microbial colonization of freshly ingested herbage in the rumen as well as fatty acid biohydrogenation. In the first study, fresh perennial ryegrass was subjected to a range of sample preparation methods before incubation in the rumen for 2 or 7 h. In the second study, fresh perennial ryegrass was chopped into 1-cm lengths and incubated in polyester bags in the rumen for 2, 8, and 24 h. After removal of bags from the rumen, 4 different washing methods, ranging from manual squeezing to machine washing, were applied. Fatty acids were extracted from washed residues and determined, as methyl esters, by gas chromatography. The main odd-chain fatty acids (with the exception of anteiso C(15:0)) were not found in fresh grass and were useful markers of the effects of incubation time, sample preparation method, and washing method on microbial colonization/contamination. The concentration of these and other odd-chain fatty acids increased with incubation time in both studies. The results indicate rapid and continued microbial colonization of freshly ingested forages, although patterns of odd-chain fatty acids did not reveal any further information about the types of bacteria-colonizing herbage. Principal component, biplot analysis provided a useful overall description of the processes of microbial colonization and degradation of plant fatty acids on fresh herbage incubated in the rumen. Bolus formation during mastication and ingestion results in extensive damage to herbage; none of the techniques (cutting, crushing, and drying/grinding) investigated in this work was able to replicate the effects of bolus formation in the animal. The study provided further evidence of loss of unfermented feed particles through polyester bag pores, especially when feeds are dried and ground. Biohydrogenation of the polyunsaturated fatty acids of fresh herbage was used principally by solid-associated bacteria to enable them to take up high levels of trans-11 C(18:1) and C(18:0) fatty acids. Although trans-11 C(18:1) was strongly associated with bacterial markers (odd- and branched-chain fatty acids), its precursor (cis-9, trans-11 C(18:2)) was not associated with bacterial variation, suggesting that its production in the rumen under these conditions was mainly extracellular.

Analysis of Variance↗