Influence of supplemental endoglucanase or xylanase on volatile fatty acid production from ruminant feed by ruminal in vitro cultures

Abstract

This study employed two commercial enzyme preparations to examine the effects of endoglucanase, xylanase or their combination on in vitro volatile fatty acid (VFA) production by ruminal microbial populations. Batch ruminal cultures were established with one of various feedstuffs or with a fescue hay-based diet and ruminal fluid from a heifer fed a 40% forage:60% concentrate diet. Addition of xylanase at 135 xylanase units (XU) per ml increased total VFA production from the fescue hay-based diet (44.3 vs. 57.2 mM, p < 0.05) without changing the acetate to propionate (A:P) ratio. Addition of endoglucanase at 2, 3, 4, and 5 carboxymethyl cellulase units (CMCU) per ml increased total VFA production from the fescue hay-based diet on average by 36% (p < 0.05). Addition of 3, 4 and 5 CMCU/ml also decreased (p < 0.05) the A:P ratio. The combined addition of xylanase (135 XU/ml) and endoglucanase (5 CMCU/ml) increased total VFA production from the fescue hay-based diet (40.9 vs. 61.5 mM, p < 0.05) and reduced the A:P ratio (3.4 vs. 1.5, p < 0.05). The effects of endoglucanase and xylanase supplementation on in vitro VFA production varied across the various substrates used. However, endoglucanase supplementation consistently reduced the A:P ratio with all substrates tested. The effects of the enzyme combination were generally greater than either enzyme alone. We conclude that endoglucanase and xylanase activities differ in their ability to affect ruminal VFA production, and endoglucanase but not xylanase, may improve fermentation efficiency by reducing the A:P ratio.     

Assessment of the efficacy of varying experimental exogenous fibrolytic enzymes using in vitro fermentation characteristics

This study evaluated a series of recombinant, single activity experimental enzyme products including 13 endoglucanases (END) and 10 xylanases (XY), for their potential to improve in vitro ruminal degradation of alfalfa hay in two experiments. Based on the endoglucanase or xylanase enzymatic activities measured using complex substrates at the optimal conditions (pH 5.4, 37 °C) for the enzymes, a dose level (1 unit/g dry matter [DM]) was chosen for addition of enzymes to substrate. Enzyme products, re-suspended with water, were added to alfalfa hay (0.5 or 1.0 g DM) in culture vials in six replications. Anaerobic buffer medium (20 or 40 ml) adjusted to pH 6.0 and strained ruminal fluid (5 or 10 ml) were sequentially added to the vials and incubated for 18 h. Headspace gas production (GP) was measured throughout the incubation, and degradability of organic matter (OMD) and fibre and volatile fatty acid (VFA) concentrations were determined after 18 h of incubation. The enzyme products had a wide range of added endoglucanase or xylanase activities when determined using pure substrates and physiological conditions typical of the rumen (pH 6.0, 39 °C). In experiment 1, many END, and some XY, products increased GP and OMD. The correlation between added endoglucanase activity determined at ruminal conditions and OMD improvement was high (r = 0.71; P<0.01), whereas added activity of xylanase was not associated with OMD improvement. Two END and two XY products selected from experiment 1 were further assessed because they substantially improved GP and OMD. In experiment 2, all enzyme treatments, alone or in combination, increased total GP and DM and fibre degradabilities (P<0.05). However, the combinations of END and XY did not increase degradation of alfalfa beyond that of the component enzymes. Total VFA production was not affected by enzyme treatments although some products changed the acetate to propionate ratio. Experimental exogenous enzyme products with either endoglucanase or xylanase activity substantially improved in vitro ruminal degradation of alfalfa hay, but further improvement by combining these activities did not occur.     

Effects of exogenous fibrolytic enzymes on in vitro ruminal fermentation of substrates with different forage:concentrate ratios

Batch cultures of mixed rumen micro-organisms were used to study the effects of three fibrolytic enzymes (xylanase from Trichoderma viride (XYL) and fibrolytic enzymes from Aspergillus niger (ASP) and Trichoderma longibrachiatum (TR)) on the fermentation of three substrates composed of grass hay:concentrate in the proportions (dry matter (DM) basis) of 0.7:0.3 (HF), 0.5:0.5 (MF) and 0.3:0.7 (LF). Enzymes were characterized for xylanase, endoglucanase, exoglucanase and amylase activities, and were supplied at rates of 40 and 80 enzymatic units/g substrate DM. In 8 h incubations, all enzymes increased (P=0.048 to P<0.001) the true degradability of substrate DM and the production of acetate, propionate, total volatile fatty acids (VFA) and gas. After 24 h incubation, some of the observed effects disappeared, but all enzymes still increased (P=0.028 to P<0.001) the degradability of substrate acid detergent fibre and the production of acetate, propionate and total VFA. For all enzymes, the effects on ruminal variables were less marked at 24 than at 8 h of incubation. Only few significant (P=0.044 to P=0.001) enzyme × substrate interactions were detected, although the magnitude of the response for each substrate varied with the enzyme. When considering the amount of organic matter apparently fermented (OMAF) and the methane:OMAF ratio as main variables, TR80 produced the greatest increase in OMAF (17.0%) for HF substrate, with ASP80 and TR40 having similar values (11.1 and 12.6%), and XYL and ASP40 showing no effects (P>0.05). A decrease (P<0.05) of methane:OMAF ratio was only found for TR80 at 8 h (17.4%). All enzymes, with the exception of ASP40, increased (P<0.05) OMAF at 8 h for MF substrate (11.3–25.4%), TR80 showing the greatest response. After 24 h of incubation, both doses of XYL and TR increased (P<0.05) OMAF (mean value 8.2%) and decreased methane:OMAF ratio (mean value 9.5%). All enzymes increased significantly OMAF with LF substrate at 8 h (7.5–19.9%), but after 24 h no effect (P>0.05) was detected on OMAF and methane:OMAF ratio. In general, few differences were detected between both doses of enzymes, which indicate than the used enzymes would be effective in enhancing ruminal degradation of substrates at a dose lower than 80 enzymatic units/g substrate DM.     

Effects of varying proportions of concentrates on ruminal-reducing power and bacterial community structure in dry dairy cows fed hay-based diets

The objectives of this study were to evaluate the influence of diet composition on ruminal parameters, more particularly redox potential (Eh). Four Holstein dry dairy cows, fitted with ruminal cannulas, were allocated in a 4 × 4 Latin square design. They were given four experimental hay-based diets D0, D25, D42 and D56 consisting of 0%, 25%, 42% and 56% of ground wheat and barley concentrate mixture, respectively. They were fed at a daily feeding rate of 8.0 kg DM per cow during a 24-day experimental period (a 21-day diet adaptation, three consecutive days of measurement and sampling). The physicochemical parameters, such as pH and Eh, were measured and Clark's exponent (rH) was calculated from 1 h before feeding to 8 h after feeding at 1-h interval. Samples of ruminal fluid were taken at 0, 1, 2, 4, 6 and 8 h after feeding for the determination of volatile fatty acid (VFA) and ammonia N (NH3-N) concentrations. Ruminal bacterial populations were also studied by means of capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) technique to focus on the structure of the ruminal microbiota and the diversity index was calculated. Mean ruminal Eh and rH were not modified by the concentrate-to-forage ratio and averaged - 210 mV and 6.30, respectively, across diets. The pH decreased slightly by 0.10 pH unit between treatments D0 and D56 with an average of 6.58. Nevertheless, the time during which physicochemical parameters remained at nadir value after feeding varied with diets: 2 and 7 h for D0 and 6 and 5 h for D56, respectively for pH and Eh. Moreover, fermentative parameters were altered by treatments: total VFA and NH3-N were greater in D56 (72.2 mM and 17.5 mg/100 ml) compared with D0 (65.2 mM and 14.2 mg/100 ml). However, neither the structure of bacterial populations of the rumen nor the diversity index (Shannon) was altered by treatments.     

Production of extracellular enzymes by immobilizedTrichoderma reesei in shake flask cultures

Polyurethane foam and nylon-web carriers were compared for simultaneous production of endo-1, 4-β-glucanase and xylanase by immobilizedTrichoderma reesei on a medium based on lactose (27 g/l), cellulose (3 g/l) and sorbose (0.3 g/l). Nylon sheet with 1.2 cm² carrier surface/ml medium was superior to the others, and it was selected for further studies. The carbon source had a marked effect on enzyme production by the immobilized fungus. With pure cellulose (10 g/l) as substrate, the maximum endoglucanase activity was 690 nkat/ml and xylanase activity 4800 nkat/ml. Supplementation with 0.5 g/l of sorbose resulted in an increase in both endoglucanase and xylanase activities in all media studied. A more detailed study on the effect of sorbose on a lactose(7 g/l)-and cellulose(3 g/l)-based medium revealed a clear optimum sorbose concentration of 1.5 g/l, with a maximum endoglucanase activity of 660 nkat/ml, xylanase activity of 3670 nkat/ml, and filter-paper activity (overall cellulolytic activity) of 2.0 filter-paper units/ml. However, the addition of 1.5 g/l sorbose to the pure-cellulose(10 g/l)-based medium resulted in a slight decrease in the enzyme production.     

The effects of xylanase on grower pig performance,concentrations of volatile fatty acids and peptide YY in portal and peripheral blood

Non-starch polysaccharides (NSP) present in wheat and barley can act as anti-nutrients leading to an increase in digesta viscosity and a reduction in nutrient digestibility. Xylanase, an NSP-degrading enzyme, has been shown to increase nutrient digestibility in pigs. The objectives of this study were: (1) to identify the optimum inclusion level of xylanase in grower pig diets by measuring the effect of increasing enzyme levels on growth performance, the concentration of volatile fatty acids (VFA) and peptide YY concentration in portal and peripheral blood of grower pigs and (2) to increase our understanding of the interrelationships between xylanase inclusion, VFA production and peptide YY secretion. A total of 512 grower pigs ((Large White×Landrace)×MAXGRO) were allocated to pens creating 32 replicates of four pigs per pen per treatment. Pigs were allocated to trial weighing 14.2±0.31 kg and remained on trial until ~41.5±3.31 kg. The experiment was a dose response design with four inclusion levels (0, 8000, 16 000 or 32 000 BXU/kg) of xylanase (Econase XT). Diets were cereal-based wheat, barley mix formulated to meet or exceed the nutrient requirements of grower pigs. Body weight and feed intake were recorded to calculate growth performance. Pen faecal samples were collected to estimate DM, organic matter (OM) and crude fibre (CF) apparent total-tract digestibility. At the end of the trial 16 pigs per treatment were euthanised by schedule 1 procedures. Peripheral and portal blood samples were collected for peptide YY and VFA analysis. The addition of xylanase to the diet had no effect on growth performance, DM, OM or CF total-tract digestibility; however, xylanase tended to have a quadratic effect on ileum pH with higher pH values recorded for pigs fed a diet supplemented with 8000 and 16 000 BXU/kg xylanase (P<0.1). Xylanase had no effect on peptide YY levels or VFA concentration. Total VFA concentration was higher in portal compared with peripheral blood (P<0.05). In conclusion, the addition of xylanase had no effect on grower pig performance, nutrient digestibility, VFA concentration or peptide YY concentration when fed up to 32 000 BXU/kg over a 35-day period. Pig performance was good for all treatments throughout the trial suggesting that diet quality was sufficient thus there were no beneficial effects of adding xylanase.     

Is the anthelmintic effect of heather supplementation to grazing goats always accompanied by anti-nutritional effects?

To test the hypothesis that the beneficial anthelmintic effect of consuming moderate amounts of tannins may not always be accompanied by anti-nutritional effects in goats, two experiments were conducted. In the first, 48 Cashmere goats were randomly assigned to two treatments: supplementation with tannin-containing heather (6.4% total tannins) and non-supplementation. All goats grazed continuously from May to September under farm conditions in a mountainous area of northern Spain. The mean percentage of heather incorporated into the diet of the supplemented animals was 29.1%. Supplementation reduced the mean number of nematode eggs in faeces (P < 0.001) and the goat mortality rate (P < 0.05). The rumen ammonia concentration was markedly reduced in the goats receiving the heather supplement (160 v. 209 mg/l; P < 0.01), while volatile fatty acid (VFA) concentrations were significantly greater (63.0 v. 53.6 mmol total VFA/l; P < 0.05). The heather-supplemented goats also showed a lower loss of live weight (P < 0.01) and body condition score (P < 0.001). In the second experiment, batch cultures of rumen microorganisms with rumen fluid from nine goats whose diet included 29% heather - or not, were used to incubate three substrates (pasture, pasture + heather and pasture + heather + polyethylene glycol) to investigate in vitro ruminal fermentation. Differences (P < 0.01) among substrates were observed in terms of dry matter disappearance (DMD), in vitro true substrate digestibility (ivTSD), gas production and ammonia concentration, the greatest values always associated with the pasture substrate. Cultures involving rumen inoculum derived from goats receiving the heather-containing diet showed slightly lower DMD (46.9 v. 48.5 g/100 g; P < 0.05), ivTSD (64.6 v. 65.9 g/100 g; P < 0.10) and gas production (105 v. 118 ml/g; P < 0.001) values, but much greater total VFA concentrations (48.5 v. 39.3 mmol/l; P < 0.05), and suggest that the efficiency of ruminal fermentation in these animals was probably improved. Together, the results support the absence of a clear nutritional cost counteracting the beneficial anthelmintic effect of supplementing the diet of grazing goats with tannin-containing heather.     

Enhanced xylanase and endoglucanase production from Beauveria bassiana SAN01, an entomopathogenic fungal endophyte

This study was undertaken to explore alternative applications of the widely known entomopathogenic/endophytic fungus, Beauveria bassiana, besides its sole use as a biocontrol agent. B. bassiana SAN01, was investigated for the production of two glycoside hydrolases, xylanase and endoglucanase under submerged conditions. Among the different biomass tested, wheat bran provided the best results for both xylanase and endoglucanase, and their production levels were further enhanced using response surface methodology. Under optimised conditions, heightened yields of 1061 U/ml and 23.03 U/ml were observed for xylanase and endoglucanase, respectively, which were 3.44 and 1.35 folds higher than their initial yields. These are the highest ever production levels reported for xylanase and endoglucanase from any B. bassiana strain or any known entomopathogenic fungi. Furthermore, the efficacy of xylanase/endoglucanase cocktail in the saccharification of sugarcane bagasse was evaluated. The highest amount of reducing sugar released from the pretreated biomass by the action of the crude Beauveria enzyme cocktail was recorded at 30°C after 8 h incubation. The significant activities of the hydrolytic enzymes recorded with B. bassiana in this study thus present promising avenues for the use of the entomopathogen as a new source of industrial enzymes and by extension, other biotechnological applications.     

Effects of fibre level and dietary mannanoligosaccharides on digestibility,caecal volatile fatty acids and performances of growing rabbits

The current experiment with 3 trials aimed to study the effect of two levels of dietary fibre – high fibre (HF; 323 g aNDFom/kg) and low fibre (LF; 248 g aNDFom/kg) – and the effect of mannanoligosaccharides (MOS) addition (1 g/kg) to the LF diet (LFM) on the performances and health status of growing rabbits, digestibility and caecal fermentative characteristics. In the growth trial 132 rabbits of both sexes were used (11 cages with 4 rabbits per treatment) from weaning (32 days of age) to slaughter (67 days of age). Rabbits fed HF diet showed a significantly higher weight gain and live weight at 67 days than rabbits fed LF diet (2032 g vs. 1935 g) (P<0.05). Feed and digestible energy intake increased with dietary fibre level (P<0.05). During the growing period rabbits fed HF diet had a feed intake 26% higher than those fed LF diet. Feed efficiency ratio was worse in HF animals (0.334 vs. 0.385; P<0.05). Addition of MOS to LF diet did not affect growth performance parameters (P>0.05). Mortality and morbidity rate were not affected by treatments. In the digestibility 24 rabbits from 46 to 51 days of age trial were used. The HF diet resulted in a significant (P<0.05) decrease in digestibility of dry matter, organic matter and protein while the aNDFom digestibility was not significantly different between diets (P>0.05). Supplementation with MOS had no effects on digestibility (P>0.05). In the 3rd trial the caecal traits were measured in 30 rabbits with 46 days of age that received the experimental diets in the previous 14 days. Caecal production of total volatile fatty acids (VFA), acetate and propionate were significantly higher (P>0.05) on rabbits fed HF diets than on rabbits fed LF diets. The total VFA concentration increased 64% (from 5.01 to 8.20 mmol/100 ml) and acetate increased 73% (from 3.73 to 6.44 mmol/100 ml). Butyrate production was not different between diets (P>0.05). Fibre level did not affect proportions of VFA and caecal contents and caecal weights. Addition of MOS to LF diet did not affect any caecal trait (P>0.05). It was concluded that the reduction of dietary fibre level increases feed digestibility but worsens rabbit growth performances. Supplementation of low fibre diet with 1 g MOS/kg is not enough to reduce its negative effects on growth performances.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Effects of soybean oil and dietary copper levels on nutrient digestion,ruminal fermentation,enzyme activity,microflora and microbial protein synthesis in dairy bulls

ABSTRACT

The study evaluated the effects of soybean oil (SO) and dietary copper levels on nutrient digestion, ruminal fermentation, enzyme activity, microflora and microbial protein synthesis in dairy bulls. Eight Holstein rumen-cannulated bulls (14 ± 0.2 months of age and 326 ± 8.9 kg of body weight) were allocated into a replicated 4 × 4 Latin square design in a 2 × 2 factorial arrangement with factors being 0 or 40 g/kg dietary dry matter (DM) of SO and 0 or 7.68 mg/kg DM of Cu from copper sulphate (CS). The basal diet contained per kg DM 500 g of corn silage, 500 g of concentrate, 28 g of ether extract (EE) and 7.5 mg of Cu. The SO × CS interaction was significant (p < 0.05) for ruminal propionate proportion and acetate to propionate ratio. Dietary SO addition increased (p < 0.05) intake and total tract digestibility of EE but did not affect average daily gain (ADG) of bulls. Dietary CS addition did not affect nutrient intake but increased (p < 0.05) ADG and total tract digestibility of DM, organic matter, crude protein and neutral detergent fibre. Ruminal pH was not affected by treatments. Dietary SO addition did not affect ruminal total volatile fatty acids (VFA) concentration, decreased (p < 0.05) acetate proportion and ammonia N and increased (p < 0.05) propionate proportion. Dietary CS addition did not affect ammonia N, increased (p < 0.05) total VFA concentration and acetate proportion and decreased (p < 0.05) propionate proportion. Acetate to propionate ratio decreased (p < 0.05) with SO addition and increased (p < 0.05) with CS addition. Dietary SO addition decreased (p < 0.05) activity of carboxymethyl cellulase, cellobiase and xylanase as well as population of fungi, protozoa, methanogens, Ruminococcus albus and R. flavefaciens but increased (p < 0.05) α-amylase activity and population of Prevotella ruminicola and Ruminobacter amylophilus. Dietary CS addition increased (p < 0.05) activity of cellulolytic enzyme and protease as well as population of total bacteria, fungi, protozoa, methanogens, primary cellulolytic and proteolytic bacteria. Microbial protein synthesis was unchanged with SO addition but increased (p < 0.05) with CS addition. The results indicated that the addition of CS promoted nutrient digestion and ruminal fermentation by stimulating microbial growth and enzyme activity but did not relieve the negative effects of SO addition on ruminal fermentation in dairy bulls.     

Engineering of a multifunctional hemicellulase

To engineer a multifunctional xylan-degrading enzyme, a chimera was created by fusing the xylanase domain of the Clostridium thermocellum xylanase (xynZ) and a dual functional arabinofuranosidase/xylosidase (DeAFc; from a compost starter mixture) through a flexible peptide linker. The xylanase domain of xynZ possesses previously unreported endoglucanase activity. The chimera, possessing the activities of xylanase, endoglucanase, arabinofuranosidase and xylosidase, was expressed in E. coli and purified. The chimera closely resembled the parental enzymes in pH, temperature optima and kinetics, and was more active than the parental enzyme mixture in the hydrolysis of natural xylans and corn stover. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Xylanase production by Trichoderma harzianum E58

Summary Growth of Trichoderma harzianum E58 on hemicellulose-rich media, both in batch and fermentor cultures, resulted in independent profiles for the production of xylanase and endoglucanase enzymes. Dramatic differences in the ratio of xylanase to endoglucanase activities were observed among cultures grown on cellulose-rich Solka Floc and xylan. These results indicated that the induction of xylanases and cellulases was likely to be under separate regulatory control. The specific activity and amount of xylanases produced were found to be dependent on the concentration of xylan in the growth media. Growth on oat spelts xylan or the hemicellulose-rich, water-soluble fraction from steam-treated aspenwood (SEA-WS) greatly enhanced the production of xylanases and xylosidase in the culture filtrates. Constitutive levels of xylanase and endoglucanase enzymes were detected during growth of the fungus on glucose.Offprint requests to: D. J. Senior     

Soybean oil and linseed oil supplementation affect profiles of ruminal microorganisms in dairy cows

The objective of this study was to evaluate changes in ruminal microorganisms and fermentation parameters due to dietary supplementation of soybean and linseed oil alone or in combination. Four dietary treatments were tested in a Latin square designed experiment using four primiparous rumen-cannulated dairy cows. Treatments were control (C, 60 : 40 forage to concentrate) or C with 4% soybean oil (S), 4% linseed oil (L) or 2% soybean oil plus 2% linseed oil (SL) in a 4 × 4 Latin square with four periods of 21 days. Forage and concentrate mixtures were fed at 0800 and 2000 h daily. Ruminal fluid was collected every 2 h over a 12-h period on day 19 of each experimental period and pH was measured immediately. Samples were prepared for analyses of concentrations of volatile fatty acids (VFA) by GLC and ammonia. Counts of total and individual bacterial groups (cellulolytic, proteolytic, amylolytic bacteria and total viable bacteria) were performed using the roll-tube technique, and protozoa counts were measured via microscopy in ruminal fluid collected at 0, 4 and 8 h after the morning feeding. Content of ruminal digesta was obtained via the rumen cannula before the morning feeding and used immediately for DNA extraction and quantity of specific bacterial species was obtained using real- time PCR. Ruminal pH did not differ but total VFA (110 v. 105 mmol/l) were lower (P < 0.05) with oil supplementation compared with C. Concentration of ruminal NH3-N (4.4 v. 5.6 mmol/l) was greater (P < 0.05) due to oil compared with C. Compared with C, oil supplementation resulted in lower (P < 0.05) cellulolytic bacteria (3.25 × 108 v. 4.66 × 108 colony-forming units (CFU)/ml) and protozoa (9.04 × 104 v. 12.92 × 104 cell/ml) colony counts. Proteolytic bacteria (7.01 × 108 v. 6.08 × 108 CFU/ml) counts, however, were greater in response to oil compared with C (P < 0.05). Among oil treatments, the amount of Butyrivibrio fibrisolvens, Fibrobacter succinogenes and Ruminococcus flavefaciens in ruminal fluid was substantially lower (P < 0.05) when L was included. Compared to C, the amount of Ruminococcus albus decreased by an average of 40% regardless of oil level or type. Overall, the results indicate that some ruminal microorganisms, except proteolytic bacteria, are highly susceptible to dietary unsaturated fatty acids supplementation, particularly when linolenic acid rich oils were fed. Dietary oil effects on ruminal fermentation parameters seemed associated with the profile of ruminal microorganisms.     

Optimisation of culture medium and conditions for alpha-l-Arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus

The culture medium for Rhodothermus marinus was optimised on a shake-flask scale by using statistical factorial designs for enhanced production of a highly thermostable alpha-L-arabinofuranosidase (AFase). The medium containing 3.6 g/l birch wood xylan and 8.2 g/l yeast extract yielded a maximum of 110 nkat/ml AFase activity together with 125 nkat/ml xylanase and 65 nkat/ml beta-xylosidase activity. In addition, low levels of beta-mannanase (30 nkat/ml), alpha-galactosidase (0.2 nkat/ml), beta-galactosidase (0.3 nkat/ml), endoglucanase (5 nkat/ml) and beta-glucosidase (30 nkat/ml) were detected in the culture filtrate. Among the various carbon sources tested, birchwood xylan was most effective for the formation of AFase and xylanase activities, followed by oat spelt and beechwood xylans, and xylan-rich lignocelluoses (e.g., starch-free sugar beet pulp and wheat bran). Constitutive levels of enzyme activities were detected when the bacterium was grown on other polysaccharides and low-molecular-weight carbohydrates. A fermentation in a 5-l fermenter (3-l working volume) using the optimised medium yielded 60 nkat/ml AFase associated with 65 nkat/ml xylanase and 35 nkat/ml beta-xylosidase activities. The crude AFase displayed optimal activity between pH 5.5 and 7 and at 85 degrees C. It had half-lives of 8.3 h at 85 degrees C and 17 min at 90 degrees C. It showed high stability between pH 5 and 9 (24 h at 65 degrees C). The combined use of AFase-rich xylanase and mannanase from R. marinus in the prebleaching of softwood kraft pulp gave a brightness increase of 1.8% ISO. To our knowledge, this is the first report on the production of a high AFase activity by an extreme thermophilic bacterium and this enzyme is the most thermostable AFase reported so far.     

Effect of 2-bromoethanesulfonic acid and Peptostreptococcus productus ATCC 35244 addition on stimulation of reductive acetogenesis in the ruminal ecosystem by selective inhibition of methanogenesis.

Evidence is provided that reductive acetogenesis can be stimulated in ruminal samples during short-term (24-h) incubations when methanogenesis is inhibited selectively. While addition of the reductive acetogen Peptostreptococcus productus ATCC 35244 alone had no significant influence on CH4 and volatile fatty acid (VFA) production in ruminal samples, the addition of this strain together with 2-bromoethanesulfonic acid (BES) (final concentration, 0.01 or 0.03 mM) resulted in stimulation of acetic acid production and H2 consumption. Since acetate production exceeded amounts that could be attributed to reductive acetogenesis, as measured by H2 consumption, it was found that P. productus also fermented C6 units (glucose and fructose) heterotrophically to mainly acetate (> 99% of the total VFA). Using 14CH3COOH, we concluded that addition of BES and BES plus P. productus did not alter the consumption of acetate in ruminal samples. The addition of P. productus to BES-treated ruminal samples caused supplemental inhibition of CH4 production and stimulation of VFA production, representing a possible energy gain of about 13 to 15%.     

Carbohydrate-hydrolyzing enzyme ratios during fungal degradation of woody and non-woody lignocellulose substrates

Understanding the order that enzymes are secreted during lignocellulosic degradation is relevant both to better understanding basic fungal degradation mechanisms and to industrial attempts to control reactions for biofuels production and other bioprocessing technology. Much is known about the enzymes that are produced and their effect on individual substrates, but little is known about temporal variation and relative enzyme activity on different lignocellulosics substrates. Wood decay fungi Trametes versicolor and Postia placenta were grown in liquid culture with different substrates (aspen, pine, corn stover, prairie grass and alfalfa) over a 16-week period. Samples of liquid media were taken every 2 weeks for endoglucanase, β-glucosidase and xylanase activity measurement. Endoglucanase:β-glucosidase:xylanase ratios varied for both fungi over the sampling period. T. versicolor showed large differences in cellulase enzyme (total cellulase:endoglucanase:β-glucosidase) composition when grown on woody substrates compared with non-woody substrates; there were also difference between the two wood types. This research presents evidence that the ratio of carbohydrate-hydrolyzing enzymes secreted by fungi is not influenced solely by lignin:carbohydrate content of the substrate and other factors including cell anatomy and constituent composition have some control on enzyme production. This provides a useful and broad survey of natural adaptations to various plant tissues relevant to bioenergy and general bioprospecting.     

Effects of Yucca schidigera and Quillaja saponaria with or without β 1–4 galacto-oligosaccharides on ruminal fermentation,methane production and nitrogen utilization in sheep

Effects of Quillaja saponaria extract (QSE) and Yucca schidigera extract (YSE) with or without β 1–4 galacto-oligosaccharides (GOS) on ruminal fermentation, methane production and N utilization in wether sheep were evaluated. Four wethers fitted with permanent ruminal fistulae were assigned in a 4 × 6 Youden square design experiment and fed a basal diet comprised of concentrate and Italian ryegrass hay (2:3, on a DM basis) at 55 g/kg metabolic body weight. Treatments were: (1) control (no addition of supplement); (2) 14 ml of QSE; (3) 14 ml of YSE; (4) 20 g of GOS; (5) 14 ml QSE + 20 g GOS; (6) 14 ml YSE + 20 GOS per day. Digestibility of NDFom increased (P<0.05) in sheep treated with QSE, QSE + GOS, and YSE + GOS, but was not affected by YSE or GOS. Ruminal fluid pH increased (P<0.05) in sheep administered with YSE compared with other treatments. Ammonia N and total VFA concentrations declined (P<0.001) with administration of QSE and YSE compared with the control. Propionate and acetate molar proportions were not affected, while butyrate molar proportion declined (P<0.001) with QSE alone or in combination with GOS. Digestible energy increased (P<0.05) with all treatments, except YSE. Results suggest that administration of QSE and YSE reduced ruminal ammonia N and total VFA concentration, and numerically decreased methane emissions, without having adverse effects on fiber digestion. In addition, administration of YSE, with or without GOS, numerically reduced protozoal numbers. QSE, YSE and YSE + GOS may have potential as beneficial manipulators of ruminal fermentation.     

Electrophoretic and enzymatic studies on the crude extracellular enzyme system of the cellulolytic bacterium Cellulomonas sp. ATCC 21399

Cellulomonas sp. ATCC 21399 produced extracellular enzyme activities against Avicel, H(3)PO(4)-swollen Avicel, carboxymethylcellulose, (1-3, 1-4)-beta-D-heteroglucan, xylan, galactomannan, and amylose drying growth on microcrystalline cellulose. No extracellular cellobiase activity was produced. Crossed immunoelectrophoresis of the crude extracellular enzyme system revealed 15 immunologically distinct immunoprecipitates. The immunoprecipitates of endoglucanase A, endoglucanase B and the xylanase appeared heterogeneous with several optima, whereas the immunoprecipitates of endoglucanase C and the amylase appeared homogeneous. The heterogeneity of endoglucanase A, endoglucanase B and xylanase was also visualized using electrofocusing-immunoelectrophoresis. Electro-focusing could resolve the activity against carboxymethylcellulose into six peaks, whereas only one peak of activity against Avicel was observed. The later peak coincided with the major peak of activity against carboxymethylcellulose with isoelectric point between pH 4.0-5.0.