Bacteriocin-Like Activity of Butyrivibrio fibrisolvens JL5 and Its Effect on Other Ruminal Bacteria and Ammonia Production

When ruminal bacteria from a cow fed hay were serially diluted into an anaerobic medium that had only peptides and amino acids as energy sources, little growth or ammonia production was detected at dilutions greater than 10⁻⁶. The 10⁻⁸ and 10⁻⁹ dilutions contained bacteria that fermented carbohydrates, and some of these bacteria inhibited Clostridium sticklandii SR, an obligate amino acid-fermenting bacterium. Phylogenetic analysis indicated that the most active isolate (JL5) was closely related to Butyrivibrio fibrisolvens B835. Strain JL5 inhibited B. fibrisolvens 49 and a variety of other gram-positive organisms, but it had little effect on most gram-negative ruminal bacteria. Strain JL5 did not produce a bacteriocin-like inhibitory substance (BLIS) until it reached the late log or stationary phase. The JL5 BLIS did not cause the lysis of B. fibrisolvens 49, but the intracellular potassium level, the ATP level, the electrical potential, and the viability decreased rapidly. The JL5 BLIS also caused marked decreases in the viability and cellular potassium level of C. sticklandii SR. The membrane potential and intracellular ATP level also declined. The BLIS was degraded very slowly by pronase E, but it could be precipitated with 60% ammonium sulfate and dialyzed (3,500-Da cutoff). The BLIS could be separated from other peptides by polyacrylamide gel electrophoresis, and C. sticklandii SR overlays indicated that the molecular size of this compound was approximately 3,600 Da. Based on these results, it appeared that the JL5 BLIS was a pore-forming peptide. Because carbohydrate-fermenting ruminal bacteria could inhibit the growth of obligate amino acid-fermenting bacteria, BLIS may play a role in regulating ammonia production in vivo.     

Establishment and Development of Ruminal Hydrogenotrophs in Methanogen-Free Lambs

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10⁷ to 10⁸ cells g⁻¹) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate- and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H₂ utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H₂ utilization was similar to that in conventional lambs. H₂ utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H₂ utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H₂ in the rumen.     

Bacterial and Fungal Numbers in Ruminal and Cecal Contents of the Blue Duiker (Cephalophus monticola)

Total and cellulolytic bacterial and fungal numbers were determined in ruminal and cecal contents of 20 blue duikers (Cephalophus monticola). The animals were equally divided by sex and fed two diets, either high roughage or high concentrate. The mean concentration for total bacterial numbers in the rumen was 26.0 × 10⁸/g of contents, with values ranging from 2 × 10⁸/g to 93 × 10⁸/g. Cellulolytic numbers averaged 6.0 × 10⁸/g with a range of 1.5 × 10⁸/g to 24.0 × 10⁸/g. No differences related to sex or diet were found. In contrast, total bacterial numbers in the cecum differed between diets (P < 0.02), i.e., 1,046 × 10⁶ bacteria per g for animals fed the high-forage diet compared with 166 × 10⁶/g for those fed the high-concentrate diet. Cellulolytic bacterial counts in the cecal contents averaged 3.1 and 7.0% of the total counts for the high-forage and high-concentrate diets, respectively. Low concentrations of fungi were found in both ruminal and cecal contents of some, but not all, animals. Unexpectedly, concentrations of bacteria and fungi in the rumen and cecum were highly correlated with their total numbers (concentration multiplied by total weight of contents).     

Cell suspension cultures ofOnonis natrix L. subspeciesramosissima: Establishment and culture conditions

Summary Explants from petioles, folioles or hypocotyls ofOnonis natrix have been used for calli initiation. Hypocotyls inoculated on MS medium supplemented with 2% sucrose and 0.5 mg.1^–1 2,4-D / 1 mg.1^–1 Kin showed to be the best primary explant. Cell suspension cultures were established in MS basal medium supplemented with 2% sucrose, 0.5 mg.1^–1 NAA or 2,4-D and 1 mg.1^–1 Kin. Different subculturing periods, inoculum density, hormonal supplementation and sucrose concentration were assayed in order to obtain the best culture growth conditions. The optimal conditions were achieved with cultures initiated with 40 g.1^–1 of initial inoculum, growing in MS basal medium supplemented with 4% sucrose, 0.5 mg.1^–1 NAA and 1 mg.1^–1 Kin subcultured every twelve days. Under these experimental conditions, the cultures showed a doubling time of 36.3 hours.     

Amino Acid Deamination by Ruminal <Emphasis Type="Italic">Megasphaera elsdenii</Emphasis> Strains

When ruminal fluid from a cow fed timothy hay was serially diluted (10-fold increments into anaerobic broth containing 15 mg ml⁻¹ Trypticase), the low dilutions (≤10⁻⁶) had optical densities greater than 2.0 and ammonia concentrations greater than 100 mM. The optical densities and ammonia concentrations of the 10⁻⁸ and 10⁻⁹ dilutions were very low, but large cocci were observed in the 10⁻⁸ dilution. The large cocci were isolated and identified by 16S rDNA sequencing as Megasphaera elsdenii. The freshly isolated strain (JL1) grew well on Trypticase, but less than 4% of the amino acid nitrogen in Trypticase was converted to ammonia. Optical density and ammonia production were twice as great if Casamino acids were provided, and similar results were obtained with seven other strains (B159, AW106, YT91, LC1, T81, J1, and YZ70). Specific activities of deamination (based on Casamino acids) of the eight strains ranged from 100 (strain JL1) to 325 (strain B159) nmol mg protein⁻¹ min⁻¹. None of the strains could utilize branched-chain amino acids as an energy source for growth, but specific activities of branched-chain amino acid deamination ranged from 15 to 65 nmol mg protein⁻¹ min⁻¹. All eight of the M. elsdenii strains grew well in the presence of 5 μM monensin, and only two of the strains were strongly inhibited by 20 μM monensin. On the basis of these results, it appears that M. elsdenii is deficient in peptidase activity and can utilize only a few amino acids. Some M. elsdenii strains produced ammonia and branched-chain volatile fatty acids nearly as fast as obligate amino acid-fermenting ruminal bacteria, but the extent of this production was at least fourfold lower. Because all of the strains could tolerate 5 μM monensin, it is unlikely that this feed additive would significantly inhibit M. elsdenii in vivo. Received: 12 December 2001 / Accepted: 5 February 2002     

Rapid in vitro differentiation of Sesbania bispinosa plants — a leguminous shrub

Multiple shoots differentiated from hypocotyl explants of Sesbania bispinosa (Jacq.) W.F. Wight, a leguminous woody shrub, when cultured on Gamborg's basal medium alone or in combination with 6-benzyl aminopurine (10^–7–10^–4 M). For cotyledonary explants 6-benzyl aminopurine (10^–6–10^–4 M) was necessary. The shoots rooted when cultured on Gamborg's basal medium containing indole-3-butyric acid (10^-5 M). Plantlets thus formed were transferred to soil where they have flowered and also set fruits.     

Influence of different co-cultivation temperatures,periods and media on <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated gene transfer

Tobacco leaf disc explants were inoculated with Agrobacterum tumefaciens strain GV2260 carrying p35S GUS-INT to determine the influence of different co-cultivation temperatures (18 – 26 °C), periods (24 – 96 h) and media (solid and liquid) on transformation efficiency. Kanamycin-resistant shoots developed on leaf discs inoculated with Agrobacterium after 4 weeks of culture initiation. Regenerated shoots were excised and rooted in the basal medium supplemented with 100 mg dm ^–3 kanamycin and 250 mg dm ^–3 augmentin. The rooted plantlets were finally transferred to compost and confirmed by GUS assay and PCR analysis. The highest transformation frequency was achieved from the explants co-cultivated with A. tumefaciens in liquid medium for 48 h at 22 or 24 °C.     

Genetic tranformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp) using Agrobacterium tumefaciens

Mature de-embryonated cotyledons with intact proximal end of Vigna unguiculata were cultured on B5 basal medium containing varying concentrations of BAP. Thirty-six percent of the explants produced shoots on B5 medium supplemented with 8× 10^–6 M BAP. Cotyledon explants were pre-incubated for 24 h, inoculated with A. tumefaciens pUCD2614 carrying pUCD2340, co-cultivated for 48 h and transferred to hygromycin-B (25 mg/l) containing shoot induction medium. Approximately 15–19% of the explants produced shoots on the selection medium. The elongated shoots were subsequently rooted on B5 basal medium containing hygromycin. The transgenic plants were later established in pots. The presence of hpt gene in the transgenic plants was confirmed by Southern blot hybridization.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - hpt hygromycin phosphotransferase - IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

The bacterial biota on crustose (nonarticulated) coralline algae from Tasmanian waters

The bacterial biota associated with the cuticle surface of healthy benthic samples of crustose nonarticulated coralline algae from the east coast of Tasmania (Australia) was examined by bacteriological cultivation and electron microscopy. In 32 samples studied, the viable count on Zobell's marine agar (supplemented with vitamins) was 3.3×10⁶ bacteria g^–1 wet wt. (range 2.9×10⁴–2.7×10⁷). Of 732 strains isolated from 16 out of 32 samples and identified to genus level,Moraxella was the predominant genus (66%). In contrast,Moraxella comprised only 11% of 217 strains isolated from benthic seawater samples collected at the same time as coralline algae. In 22 out of 32 algal samples examined by scanning electron microscopy, the total count was 1.6 × 10⁷ bacteria g^–1 wet wt. (range 5.1× 10⁶–3.8×107); the major morphotype was cocco-bacilli (80%). Several environmental factors did not significantly influence the viable count or generic distribution, or the total count or morphotypic distribution of bacteria on the cuticle. These factors included geographical site, season, storage of samples in aquarium conditions, and the presence or absence of abalone from shells that the coralline algae encrusted. The microbiota, consisting mostly of the nonmotile bacterial genusMoraxella, appeared to be highly adapted to its calcerous plant host.     

Repetitive somatic embryogenesis and plant recovery in white clover

Summary Breeding and selection was used to generate a population of white clover (Trifolium repens L.) from cultivar Osceola with a high embryogenic capacity. Somatic embryos were obtained from immature cotyledons of white clover placed onto EC6 basal medium containing 40 mg L^–1 of 2,4-D and 6% sucrose. The effects of 2,4-D at 20 and 40 mg L^–1 and of the carbohydrates, sucrose and maltose, were evaluated for their influence in the establishment of repetitive somatic embryogenesis. To determine the optimal protocol for plant recovery from somatic embryos, the effects of MS vs. EC6 basal salts, sucrose vs. maltose, B5 vitamins vs. yeast extract, and inclusion or exclusion of activated charcoal were evaluated. Repeated subculture of white clover somatic embryos on EC6 basal medium containing 6% sucrose with 2,4-D at 20 or 40 mg L^–1 effectively maintains repetitive embryogenesis. Medium containing MS salts with 6% maltose as the carbohydrate source was the most efficient for plant recovery.     

Factors affecting callus and shoot formation from in vitro cultures of Lens culinaris Medik.

The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these calli. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg l^–1 of BA and 0.186 mg l^–1 NAA+2.25 mg l^–1 BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.     

The Effect of Five Different Wetting Treatments on the Nutrient Content and Microbial Concentration in Hay for Horses

Five different hays were used to determine the effect of 5 different soaking and steaming treatments on the water soluble carbohydrate and microbial (bacteria and mould) contents of UK hay. Hays were subjected to the following 5 treatments: 1. Dry; 2. Steamed for 50 minutes in the Haygain- 600 steamer; 3. Soaked in water at 16°C for 9 hours; 4. Steamed then soaked and 5. Soaked then steamed. Post treatment hays were tested for water soluble carbohydrates, bacteria and mould contents. Differences between means were determined using ANOVA and least significant difference with hay (5), bale (3) and treatment (5) as fixed factors, thus n = 75. Protein and ash proportions were unaltered in any of the treatments. Soaked, steamed then soaked and soaked then steamed treatments were all equally effective at reducing water soluble carbohydrates, with significantly (P<0.05) lower mean contents (79–83 g/kg DM) compared with 126 and 122 g/kg dry matter (DM) for dry and steamed respectively. Steamed and soaked then steamed had significantly (P<0.05) less bacteria (1.04×10³ and 4.9×10² CFU/g DM) compared with soaked which increased CFU/g DM from 6.0×10⁴ in dry hay up to 3.5×10⁵. Mould contents CFU/g DM were significantly (P<0.05) reduced by steaming (2) and soaking then steaming (1.9) but no difference was seen between dry (1148), soaked (692) or steamed then soaked (501). Soaking for 9 hours followed by steaming for 50 minutes in the Haygain steamer was the most effective method for reducing water soluble carbohydrates and microbial contamination in hay. Soaking or steaming+soaking lowered water soluble carbohydrates but significantly reduced the hygienic quality of the hay which could potentially compromise the health of the horse.     

Cell protein degradation in cultured rat embryo fibroblasts suppression by vinblastine of the enhanced proteolysis by serum-deficient media

Rat embryo fibroblasts grown in Eagle's minimal essential medium with 10% serum were labeled with L-[¹⁴C]leucine. After a 24 h cold chase, rates of proteolysis were evaluated by measuring the appearance of trichloroacetic acid-soluble ¹⁴C in the media. Cells remaining in minimal essential medium with 10% serum (basal) showed a proteolysis rate of 1% per h, whereas cells placed in minimal essential medium alone (serum-deficient) showed a stimulation of proteolysis to 3–4% per h. This enhanced proteolysis was transitory, occuring only for the first 4–8 h after cells were placed in the serum-deficient media. Vinblastine 10⁻⁵ M inhibited the enhanced proteolysis 40% but had no effect on basal proteolysis. Control experiments showed no detectable hydrolysis of extracellular proteins, nor did vinblastine affect the rate of protein synthesis. These data suggest that basal and enhanced proteolysis have at least partially distinct mechanisms in the cell and that only enhanced proteolysis involves microtubules.     

Continuous Culture of Ruminal Microorganisms in Chemically Defined Medium: II. Culture Medium Studies

Ruminal ciliates have been grown in continuous culture in chemically defined media and in the absence of viable bacteria. Oligotrichic ruminal ciliates seem to require insoluble carbohydrates for growth; the holotrichic ciliates require soluble carbohydrates, but at low concentrations. Both groups of ciliates utilize amino acids as their principal nitrogen source when these are supplied in micromolar concentrations; at millimolar concentrations, amino acids are toxic, possibly from excessive ammonia formation arising from ciliate deaminase activity. Holotrichic ruminal ciliates are destroyed by overdeposition of amylopectin when glucose is present above 0.1% concentration in the medium. Ecological requirements of ruminal ciliates are also described.     

Regeneration of plants from primary leaves of cowpea

Plants were regenerated from the in vitro cultured explants of primary leaves of cowpea (Vigna unguiculata L. Walp). Primary leaves, including the intact petiole, were excised from three-day-old seedlings and cultured on Gamborg's B5 basal medium containing 8×10^–7 M 2,4,5-trichlorophenoxyacetic acid, 1×10^–2 M L-glutamine and 1×10^–4 M adenine sulfate. Callus formed at the petiole end. Prolific shoot regeneration occurred when this callus was transferred to B5 basal medium containing 5×10^–6 M 6-benzyl-aminopurine (BAP). Regenerated shoots rooted in growth-regulator-free B5 basal medium and were established in soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA 1-napthalene acetic acid - 2,4,5-T 2,4,5-trichloro-phenoxyacetic acid     

In vitro culture of adult and juvenile bud explants of Passiflora species

Cultivar E23, an F1 hybrid of P. edulis and P. edulis f. flavicarpa is usually propagated by shoot-tip grafting. Various media were tested to evaluate the potential of E23 for in vitro propagation. Adult tissue was difficult to culture and did not respond to media containing low (<10 µM) concentrations of growth regulators. Growth of adult buds on intact stem sections was promoted by 1 week of dark incubation on MS basal medium plus 150 µM 2iP, 200 µM adenine sulphate and 17.1 µM IAA (3 mg l^–1), and further developed into shoots on MS medium plus 4.9 µM 2iP (1 mg l^–1) and 5.7 µM IAA (1 mg l^–1). By contrast, juvenile shoots of E23, and Passiflora species: edulis f. flavicarpa, edulis, alata, caerulea, mollissima, coccinea, herbertiana and suberosa grew rapidly on MS medium plus 10 µM kinetin and 5 µM IAA. Rapid multiplication was achieved on MS plus 20 µM BA, 10 µM kinetin, 5 µM IAA, and roots initiated on MS plus 5 µM IAA.Abbreviations IAA indole-3-acetic acid - 2iP N6-iso pentenyl adenine - BA N6-benzyl adenine     

Trimethylamine and methylamine as growth substrates for rumen bacteria andMethanosarcina barkeri

Trimethylamine and methylamine were found to be used as methanogenic substrates byMethanosarcina barkeri or by bacteria found in low dilutions of rumen contents. When these substrates were used as the only added carbon and nitrogen source, up to 80% of the theoretical amount of methane production was obtained.Methanosarcina were enumerated from rumen contents at 10⁵–10⁶ bacteria/ml. Pure cultures of the various major rumen bacterial species, includingMethanobacterium ruminantium strain M1, were not able to utilize these substrates as energy and/or nitrogen sources. It is suggested that, in the rumen, trimethylamine and methylamine are primarily degraded byMethanosarcina, resulting in release of ammonia which then can be utilized by other rumen bacteria.     

Assessment of Reductive Acetogenesis with Indigenous Ruminal Bacterium Populations and Acetitomaculum ruminis

The objective of this study was to evaluate the role of reductive acetogenesis as an alternative H₂ disposal mechanism in the rumen. H₂/CO₂-supported acetogenic ruminal bacteria were enumerated by using a selective inhibitor of methanogenesis, 2-bromoethanesulfonic acid (BES). Acetogenic bacteria ranged in density from 2.5 × 10⁵ cells/ml in beef cows fed a high-forage diet to 75 cells/ml in finishing steers fed a high-grain diet. Negligible endogenous acetogenic activity was demonstrated in incubations containing ruminal contents, NaH¹³CO₃, and 100% H₂ gas phase since [U-¹³C]acetate, as measured by mass spectroscopy, did not accumulate. Enhancement of acetogenesis was observed in these incubations when methanogenesis was inhibited by BES and/or by the addition of an axenic culture of the rumen acetogen Acetitomaculum ruminis 190A4 (10⁷ CFU/ml). To assess the relative importance of population density and/or H₂ concentration for reductive acetogenesis in ruminal contents, incubations as described above were performed under a 100% N₂ gas phase. Both selective inhibition of methanogenesis and A. ruminis 190A4 fortification (>10⁵ CFU/ml) were necessary for the detection of reductive acetogenesis under H₂-limiting conditions. Under these conditions, H₂ accumulated to 4,800 ppm. In contrast, H₂ accumulated to 400 ppm in incubations with active methanogenesis (without BES). These H₂ concentrations correlated well with the pure culture H₂ threshold concentrations determined for A. ruminis 190A4 (3,830 ppm) and the ruminal methanogen 10-16B (126 ppm). The data demonstrate that ruminal methanogenic bacteria limited reductive acetogenesis by lowering the H₂ partial pressure below the level necessary for H₂ utilization by A. ruminis 190A4.     

Betaine a novel candidate for rapid induction of somatic embryogenesis in tea (Camellia sinensis (L.) O. Kuntze)

Addition of betaine to the inductionmedium significantly enhanced the rapid formation ofsomatic embryos directly without callusing from maturefresh seeds of tea within two weeks of cultureinitiation. The induction response was furtherenhanced when ABA (7.5 mgl^–1) was co-supplementedwith betaine in the induction medium. The rate ofinduction of somatic embryogenesis increased linearlywith external betaine concentration. Globular somaticembryo-like structures (embryoids) were observed in 4-week old cultures when inoculated on the inductionmedium without ABA and betaine. The positive effectof ABA on the induction process was found to bedependent on the presence of betaine in the medium. ABA alone in the medium could not bring the inductionstimulus in the explants; on the contrary, it provedinhibitory. The optimum response of ABA was observedwhen the medium was supplemented with 500 to1000 mgl^–1 of betaine. Primary somatic embryosobtained in the presence of ABA and betaine were ableto produce secondary embryos. A conversion rate of15–20% was achieved upon transfer of somatic embryosof size 3–5 mm in diameter to the basal medium consistof half strength of macro nutrients, full strength ofmicro nutrients and vitamins of MS. Medium wasfurther supplemented with 100 mg l^–1 each ofadenine hemisulfate sulphate and L-glutamine, 30 gl^–1 sucrose, gelled with 7 gl^–1 bitek agar. The plantlets regenerated by this procedure did notshow any visible abnormalities. This report for thefirst time details the potential use of betaine inplant tissue culture.