The ability of rumen ciliates, Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum, to use the murein saccharides

Murein polysaccharides may contribute to a considerable part of the dry matter of bacterial cells. Their utilization by protozoa inhabiting the rumen is, however, poorly recognized. The objective of this study was to examine the ability of three species of ciliates, i.e., Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum of digest, and ferment these saccharides. The cultivation experiments showed that the enrichment of growth medium with bacterial cell wall β-glycans increased the ciliate number (p?<?0.05). A statistically significant increase (p?<?0.01) was followed by a continuous decrease (p?<?0.01) in the percentage of individuals containing β-glycans particles after 4- and 24-h incubation of ciliates with this substrate, respectively. The enzymatic experiments confirmed the ability of the examined protozoa to digest murein. E. caudatum exhibited the highest activity (8.2 unit (U)/mg protein per min), and E. maggii, the lowest (3.0 U/mg protein per min). The production rates of volatile fatty acids by starved and fed ciliate species were 0.7 and 1.6 (E. caudatum)?pmol/ciliate cell per h, 30.5 and 42.5 (E. maggii)?pmol/ciliate cell per h, and 8.3 and 19.2 (D. affine)?pmol/ciliate cell per h (p?<?0.05).     

Suitability of different media for in vitro cultivation of the ruminal protozoa species Entodinium caudatum, Eudiplodinium maggii, and Epidinium ecaudatum

Three protozoal cultivation media were tested to determine the medium which best facilitated growth and viability of key B-type ciliates isolated from the sheep rumen. Entodinium caudatum and Eudiplodinium maggii were grown anaerobically in 50-ml flasks for 32 days in Caudatum-type (C), Kisidayova (K) or Dehority (M) medium. On day 32, in media K and M, E. caudatum cell counts were high with 5.6 × 10³ and 7.8 × 10³ mL⁻¹, respectively, and the proportion of dead cells was low with 0.6 and 1.4%, respectively. E. maggii concentrations when grown in medium M and C were 2.7 × 10³ and 2.4 × 10³ mL⁻¹, respectively, with 3.9 and 14.1% dead cells. Medium M, which favoured growth of both protozoa species, was tested again and Epidinium ecaudatum was included. Protozoa were grown for a 4-month period and samples were taken in the last two months on days 1, 7, 35 and 57. Average cell concentrations were 10.0, 0.8 and 0.5 × 10³ mL⁻¹ for E. caudatum, E. maggii, and E. ecaudatum, respectively. In conclusion, medium M would appear to be the best choice for cultivating these three species in one medium.     

Evaluation of the efficiency of metabolism of dinoflagellate phosphorus and carbon by a planktonic ciliate

The trophic transfer of nutrients through the microbial food web is a key top-down control in aquatic ecosystems which is notoriously difficult to evaluate, particularly for planktonic protists. In this study, a sensitive dual-radioactive tracer technique was developed to simultaneously assess the ingestion rate, and carbon- and phosphorus-specific assimilation efficiencies, of the marine planktonic ciliate Strobilidium neptuni feeding on the autotrophic dinoflagellate Heterocapsa triquetra. Dinoflagellate prey were simultaneously 16 h pulse labelled with NaH¹⁴CO₃ and H₃³³PO₄ before being fed to the ciliate, and radioactive labels were traced into ciliate biomass and the experimental medium, as well as being monitored in the prey cells. Rates measured in short-term (10 min) incubations, as commonly used to estimate protist uptake of fluorescently labelled prey, were approximately 6 times higher and 3–6 times more variable than rates measured in longer 3–5 h incubations. The efficiency of accumulation of prey carbon (54±9%) by ciliates was lower than that of prey phosphorus (68±3%) suggesting that the phosphorus to carbon ratio in the ciliates was 1.3 times higher than in the labelled dinoflagellate biomass. Rates of phosphorus accumulation and release were combined to reveal that ciliates consumed 3.2±0.6 dinoflagellates cell^?1 h^?1. The assessment of carbon tracer release by ciliates was less reliable due to ¹⁴CO₂ exchange between the experimental media and air. The study concludes that the dual phosphorus–carbon radioactive tracer labelling of algal prey allowed the quantification of protist herbivory and nutrient remineralisation in laboratory experiments, thereby providing a potential technique for studying planktonic microbial trophic interactions in situ.     

Rumen ciliates of domestic cattle (Bos taurus taurus) in Kastamonu,Turkey, with the description of a new species

Species composition and distribution of ciliates were investigated in the rumen contents of 25 domestic cattle (Bos taurus taurus L.) living in Kastamonu, Turkey. Forty-seven species and 37 morphotypes representing 15 genera were identified. Of them, a new species of Ostracodinium was recognized and described as Ostracodinium anatolicum n. sp. This new species has two caudal lobes. The dorsal lobe is small and rounded and the ventral lobe is triangular shaped and bent toward the dorsal side like a thick hook. Furthermore, the anterior end of the macronucleus (1/5 of the length) is bent toward the left like a hook. The density of rumen ciliates in cattle was 96.8 ± 43.3 × 10⁴ cells mL⁻¹ and the mean number of ciliate species per host was 14.2 ± 4.4. Entodinium longinucleatum, E. nanellum, E. simulans and Isotricha prostoma were the most abundant species, each with a prevalence of 88%. Entodinium chatterjeei, E. bifidum m. monospinosum, Hsiungia triciliata, Oligoisotricha bubali, Ostracodinium dogieli, O. mammosum and O. munham are new host records for cattle from Turkey.     

Colonization dynamics of biofilm-associated ciliate morphotypes at different flow velocities

The impact of flow velocity on initial ciliate colonization dynamics on surfaces were studied in the third order Ilm stream (Thuringia, Germany) at a slow flowing site (0.09 m s^?1) and two faster flowing sites (0.31 m s^?1) and in flow channels at 0.05, 0.4, and 0.8 m s^?1. At the slow flowing stream site, surfaces were rapidly colonized by ciliates with up to 60 cells cm^?2 after 24 h. In flow channels, the majority of suspended ciliates and inorganic matter accumulated at the surface within 4.5 h at 0.05 m s^?1. At 0.4 m s^?1 the increase in ciliate abundance in the biofilm was highest between 72 and 168 h at about 3 cells cm^?2 h^?1. Faster flow velocities were tolerated by vagile flattened ciliates that live in close contact to the surface. Vagile flattened and round filter feeders preferred biofilms at slow flow velocities. Addition of inorganic particles (0, 0.6, and 7.3 mg cm^?2) did not affect ciliate abundance in flow channel biofilms, but small ciliate species dominated and number of species was lowest (16 species cm^?2) in biofilms at high sediment content. Although different morphotypes dominated the communities at contrasting flow velocities, all functional groups contributed to initial biofilm communities implementing all trophic links within the microbial loop.     

Intestinal ciliate composition found in the feces of the Cypriot wild donkey,Equus asinus Linnaeus, 1758

Species composition and distribution of large intestinal ciliates was investigated in the feces from 13 Cypriot wild donkeys, free-living in the Karpaz national park, Northern Cyprus. We identified 16 ciliate genera and 22 species. This is the first report on intestinal ciliates in Cypriot wild donkeys, and no endemic species were observed. The genus Cycloposthium occurred in all animals. The mean number of ciliates was 3.0±2.5×10⁴ cells ml^?1 of feces and the mean number of ciliate species per host was 6.5±4.8. Characteristics of the wild donkey ciliates was almost identical to those reported in other equids from various regions around the world. We thus conclude that there is no pronounced geographic variation in the intestinal ciliate fauna of equids.     

Structure and seasonal dynamics of the protozoan community (heterotrophic flagellates,ciliates, amoeboid protozoa) in the plankton of a large river (River Danube,Hungary)

Seasonal dynamics of all major protozoan groups were investigated in the plankton of the River Danube, upstream of Budapest (Hungary), by bi-weekly sampling over a 1-year long period. Sixty-one heterotrophic flagellate, 14 naked amoeba, 50 testate amoeba, 4 heliozoan and 83 ciliate morphospecies were identified. The estimated abundance ranges of major groups throughout the year were as follows: heterotrophic flagellates, 0.27–7.8×10⁶ ind. l^?1; naked amoebae, max. 3300 ind. l^?1; testaceans, max. 1600 ind. l^?1; heliozoans, max. 8500 ind. l^?1; ciliates, 132–34,000 ind. l^?1. In terms of biovolume, heterotrophic flagellates dominated throughout the year (max. 0.58 mm³ l^?1), and ciliates only exceeded their biovolume in summer (max. 0.76 mm³ l^?1). Naked amoeba and heliozoan biovolume was about one, and testacean biovolume 1–3, orders of magnitude lower than that of ciliates. In winter, flagellates, mainly chrysomonads, had the highest biomass, whilst ciliates were dominated by peritrichs. In 2005 from April to July a long spring/summer peak occurred for all protozoan groups. Beside chrysomonads typical flagellates were choanoflagellates, bicosoecids and abundant microflagellates (large chrysomonads and Collodictyon). Most abundant ciliates were oligotrichs, while Phascolodon, Urotricha, Vorticella, haptorids, Suctoria, Climacostomum and Stokesia also contributed significantly to biovolume during rapid succession processes. In October and November a second high protozoan peak occurred, with flagellate dominance, and slightly different taxonomic composition.     

Predation of Salmonella enterica serovar Typhimurium by the rumen protozoon Entodinium caudatum studied in vitro by fluorescence emission

Predation of bacteria by protozoa has important implications on rumen metabolism and bacterial populations. Protozoa can also restrict the passage of pathogenic bacteria to the host’s lower gastrointestinal tract. This work aimed to evaluate the predation by Entodinium caudatum (EC) and the intraprotozoal survival of Salmonella enterica serovar Typhimurium. EC cells from a monofaunated sheep were incubated for up to 105 min with a S. enterica strain producing a green fluorescent protein. Rumen fluid from a defaunated sheep (DEF) was used as a control. Fluorescence, as an index of predation, measured in the residual (protozoal) fraction was higher in EC than in DEF. 105 min after the beginning of the incubation it was higher than 30 min after. Intracellular survival of Salmonella within EC was assessed by means of a selective medium. Amounts of Salmonella in the residual fraction were higher in EC than in DEF only after 30 min. After 105 min, each protozoa engulfed 100 Salmonella cell per min. Intraprotozoal survival of ingested Salmonella was 0.0017. Predation of S. enterica by E. caudatum occurred and increased in proportion to time, but bacterial viability inside the protozoa was lower at 105 min. This study demonstrates that fluorescence emission combined with bacterial and protozoal cultures could be a reliable method for quantifying bacterial predation and viability in vitro.     

Bromeliothrix metopoides,a boom and bust ciliate (Ciliophora,Colpodea) from tank bromeliads

We investigated the recently described colpodid ciliate Bromeliothrix metopoides in a series of laboratory experiments to reveal the environmental factors that constrain this species to its peculiar habitat, i.e. the tanks of bromeliads. Our results demonstrated that the various life stages of this ciliate (bacterivorous theronts and microstome trophonts, flagellate-feeding macrostomes) have specific demands in terms of food quality and quantity. Bromeliothrix required a high food threshold (>1.4 mg C L^?1) in order to thrive. Food quality also affected resting cyst formation of B. metopoides when the experimental containers dried out. Its maximum growth rates (μ_max = 4.71 d^?1, i.e. 6.8 doublings d^?1) belong to the highest ones recorded thus far for free-living ciliates. The pH niche of B. metopoides was relatively wide (pH ～4 to >9) under optimal food conditions. However, its high sensitivity to unfavourable environmental conditions let the population collapse within several hours. We conclude that B. metopoides is a boom and bust ciliate that is specifically adapted to its peculiar habitat but virtually unviable in other environments.     

Diversity of anaerobic fungal populations in cattle revealed by selective enrichment culture using different carbon sources

We employed most probable numbers (MPNs) enumeration of enrichment cultures, combined with the use of a range of carbon sources (glucose, cellobiose, cellulose, xylan and wheat straw), to recover and identify morphologically different groups of anaerobic fungi (monocentric rhizoidal [Neocallimastix, Piromyces spp.], polycentric rhizoidal [Anaeromyces, Orpinomyces spp.], bulbous non-rhizoidal [Caecomyces, Cyllamyces spp.]) from rumen digesta, and fresh or frozen–thawed faeces of silage-fed cattle. Highest MPN counts (>10⁶ thallus forming units [TFU] g^?1 dry matter (DM)) were obtained using wheat straw but use of other carbon sources revealed large variation in the relative abundance of the morphotypes recovered in culture. Polycentric morphotypes were overall the most abundant fungi, comprising ca. 60 % of observations and recovered most frequently with xylan and wheat straw. Bulbous morphotypes showed a reciprocal pattern of occurrence, being most frequently observed on glucose, cellobiose and cellulose. Monocentric morphotypes were surprisingly the least abundant (<10 % overall), occurring mostly on glucose and wheat straw. Freezing of faeces (?20 °C/5 weeks) and thawing prior to enrichment culture reduced MPN counts by ca. 40 % from a mean of 1.8 × 10⁵ TFU g^?1 DM, but greater relative abundance of polycentric morphotypes in frozen–thawed faeces suggested differential survival in response to environmental stresses. PCR–RFLP demonstrated the simultaneous presence of seven ribotypes in one animal, but not all ribotypes could be associated with a particular genus.     

Abundance and biomass of planktonic ciliates in the sea area around Zhangzi Island,Northern Yellow Sea

We investigated the abundance and biomass of planktonic ciliates in the sea area around Zhangzi Island, Northern Yellow Sea, from July 2009 to June 2010. Ciliates were sampled monthly from surface to bottom with a 10 m depth interval at 13 sample stations along three transects. A 1 L sample of water from each depth was collected with a 2.5 L Niskin water sampler and fixed in 1% acid Lugol’s iodine solution. Water samples were pre-concentrated using the Utermöhl method and observed using an Olympus IX51 inverted microscope at 100× or 200x. The dimensions of the ciliates were measured and the cell volume of each species was estimated using appropriate geometric shapes. The carbon:volume ratio used to calculate biomass was 0.19 pg C/μm³. Abundance and biomass of the ciliate in water column were calculated as the integral of the abundance and biomass from bottom to surface, respectively. The classification of tintinnids was based on taxonomic literature. The average abundance of non-loricate ciliates was 3066 ± 2805 ind/L, ranging from 165 ind/L (50 m depth of St. B6 in July) to 26,595 ind/L (surface of St. C1 in September). The average biomass of non-loricate ciliates was 2.88 ± 2.68 μg C/L, ranging from 0.05 μg C/L (10 m depth of St. A6 in July) to 20.51 μg C/L (surface of St. A5 in August). The average tintinnid abundance was 142 ± 273 ind/L, ranging from 0 ind/L (monthly) to 2756 ind/L (surface of St. A1 in July). The average tintinnid biomass was 0.84 ± 2.19 μg C/L, ranging from 0.00 μg C/L (every month) to 37.64 μg C/L (20 m depth of St. C5 in July). The results showed that the average abundance of total ciliates was 3208 ± 2828 ind/L, ranging from 166 ind/L (10 m depth of St. A6 in July) to 26,625 ind/L (surface of St. C1 in September); the average biomass of total ciliates was 3.73 ± 3.55 μg C/L, ranging from 0.05 μg C/L (10 m depth of St. A6 in July) to 38.29 μg C/L (20 m depth of St. C5 in July). Abundance and biomass were vertically homogeneous in February, November and December, but decreased dramatically from the surface down to the bottom in other months. 23 tintinnid species were identified, 12 of which were in genus Tintinnopsis. Tintinnid species were more abundant in February, July and August. Tintinnids occupied 6.6 ± 10.2% and 19.7 ± 23.3% of the total ciliate abundance and biomass, respectively, which increased during the warm season and at coastal stations, and decreased during the cold season and at offshore stations. Large non-loricate ciliate species were prevalent in spring, while smaller species dominated in summer and autumn. The average abundance of total ciliates in water column was 132 ± 72 × 10⁶ ind/m², with increases during spring and autumn. The average biomass of total ciliates in water column was 152.57 ± 93.10 mg C/m², with increases during spring and summer. The average abundance and biomass of total ciliates in water column were greater at offshore stations than at coastal stations during spring and autumn, and were lower during summer and winter. Non-loricate ciliates, tintinnids and total ciliates showed significant positive correlation with temperature and significant negative correlation with salinity (p < 0.01). Non-loricate ciliates and total ciliates showed significant positive correlation with Chl a concentration (p < 0.01); however, relationship between Chl a concentration and tintinnids was not significant.     

Biochemical properties of a β-mannanase and a β-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan. Its activity was optimal at pH 4.5. The K_m value with galactoglucomannan as substrate was 0.50 mg ml^?1. One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 °C and pH 8.0. Its K_m value with birchwood xylan as substrate was 1.65 mg ml^?1. Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn⁺², Fe⁺³, and Cu⁺² were strong inhibitors for the mannanase.     

Varying endophyte status and energy supplementation of fresh tall fescue in continuous culture

Eight dual-flow continuous culture vessels (700 ml) were used to compare in vitro effects of toxic, endophyte-infected (E+), endophyte-free (E−), and non-toxic, endophyte-infected (EN) Jesup tall fescue (vegetative stage) on ruminal fermentation at 4 levels (0, 150, 300, and 450 g kg⁻¹ DM) of concentrate supplementation (ground corn) for a total of 12 experimental diets in a randomized incomplete block design with 2 replicates. Each culture vessel was offered a total of 15 g DM d⁻¹. Forage was fed in four equal portions (fed at 03:00, 09:00, 15:00, and 21:00 h); and corn was fed in two equal portions (fed at 09:00 and 21:00 h). Headspace gas and liquid samples were analyzed for methane, ruminal culture pH, ammonia–N, and volatile fatty acid production. Ammonia–N output (g d⁻¹) varied by grass; EN had lower values compared to those of E+ and E−. Increasing the level of grain linearly decreased ruminal culture pH, ammonia–N, acetate production, and the acetate-to-propionate ratio, whereas propionate and butyrate production increased with higher grain supplementation. Ruminal fermentation was minimally altered by the presence of the endophyte; however, for the highest level of grain fed (450 g kg⁻¹ DM fed) the methane production pattern for all three grasses was altered. In addition to having the lowest ruminal ammonia–N accumulation, the non-toxic, endophyte-infected fescue resulted in the lowest methane production measured.     

Effects of dietary whole cottonseed and crude protein level on rumen protozoal population and fermentation parameters

In this investigation in vitro and in vivo trials were performed to determine the efficacy of a cottonseed to limit protozoal population and fermentation parameters. The composition of diets given to the different treatments were as follow: (1) control (without whole cottonseed), 16% crude protein (CP), 3.2% ether extract (E.E.); (2) 20% whole cottonseed, 16% CP, 6.5% E.E.; (3) 20% whole cottonseed, 13% CP, 6.4% E.E. and (4) 20% crushed whole cottonseed, 13% CP, 6.4% E.E. DM disappearance (DMD) and fermentation characteristics of the treatments were determined by in vitro incubation studies. In the in vivo trial, ruminal fluid was taken by rumenocentesis (3 h after feeding) on days 1, 3, 5, 7, 9, 11, 14, 21 and 28 from four sheep fed about treatment diets. The pH and protozoal counts were determined in each sample, while ammonia nitrogen and volatile fatty acid (VFA) were determined in samples taken on days 7, 14, 21 and 28. The in vitro DMD after 24 h incubation decreased (p < 0.01) with the addition of cottonseed in diets 3 and 4 and DMD after 72 h incubation was highest (p < 0.01) for the control diet. The fractional rate of gas production (c) for the control and diet 2 was higher (p < 0.05) than for the diets 3 and 4. Feeding crushed whole cottonseed decreased molar proportion of propionate (p < 0.05) and increased molar proportion of butyrate (p < 0.01). Low crude protein level increased the molar proportion of propionate (p < 0.05) and decreased molar proportion of butyrate (p < 0.05) and cellolytic protozoa population (p < 0.05). Feeding cottonseed decreased (p < 0.05) the total protozoa population from approximately 500,000 to 250,000 ml⁻¹ and Holotrich and cellulolytic protozoa disappeared from the rumen of sheep and only Entodinium sp., remained. This was associated with lower concentration of ammonia nitrogen in rumen fluid of sheep fed diets 4 (p < 0.05) and 2 (p < 0.01). It was concluded that cottonseed reduced rumen fauna and ammonia nitrogen, but had no effect on ruminal VFA while the crushed whole cottonseed decreased molar proportion of isovalerate only. In vivo molar proportion of propionate and butyrate and valerate were increase and decrease, respectively, by decreasing CP percentage in treatment diets.     

Influence of natural magnesium sources on the in vitro fermentation and protozoan population in the rumen fluid collected from sheep

The objective of this experiment was to determine the effect of two types of caustic calcinated magnesite (caustic magnesite (CM) and Agromag (AG)) upon the end products of in vitro fermentation (total gas, methane, total and individual fatty acids, and VFA) and protozoan population in the rumen fluid collected from sheep. Both magnesium additives (CM and AG) as natural products in the dose of 0.01 g were added to the fermentation bottles containing rumen inoculum from sheep and different substrates. Meadow hay (MH), wheat straw (WS), amorphous cellulose (AC) and barley grain (BG) were used as substrates and incubated with the buffered rumen fluid using an in vitro gas measuring technique during 72 h of incubation. The rumen protozoa, Entodinium spp., Trichostomatids and large Entodiniomorphids and the total protozoan concentration were counted after 24 h of incubation. The methane production was significantly decreased with CM or AG, respectively, by 58 or 62% (MH), by 65% (WS), by 52% (AC) and by 58% (BG). The total VFA concentration was significantly lower compared to control for CM plus MH, WS, AC, BG and AG plus WS. The total VFA concentration was significantly higher compared to control for AG plus AC. The effect of the both additives on ciliate population was not uniform and depended on the substrates used and protozoan type. Ciliate population was significantly increased in Entodinium spp. (AG plus BG) and Diploplastron affinae (CM or AG plus BG) compared to control. Tested additives significantly decreased population of Entodinium spp. (AG plus MH or AC), Dasytricha ruminantium (AG plus AC), Ophryoscolex c. tricoronatus, Eremoplastron dilobum and Polyplastron multivesiculatum (CM or AG plus BG). It can be concluded that both natural magnesium sources influenced rumen fermentation patterns and protozoan population in vitro depending on the type of the substrate used; therefore, the relative efficacy of individual tested additive cannot be determined from these experiments. In vivo experiments are required in future.     

Feed digestion,rumen fermentation and blood biochemical constituents in Malpura rams fed a complete feed-block diet with the inclusion of tree leaves

This experiment assayed the influence of the inclusion of dried Azardirachta indica, Albizzia lebbek or Ailanthus excelsa leaves in pearl millet stover-based complete feed block diets on feed intake, nutrient utilization, rumen fermentation characteristics, ciliate protozoa population and blood biochemical constituents in adult Malpura sheep. Complete feed blocks were formulated to have roughage-to-concentrate ratio of 70:30. Pearl millet stover (PMS) was used as basal roughage; 30 parts of pearl millet stover was replaced with dried leaves either of Azardirachta indica (NL), Albizzia lebbek (SL) or Ailanthus excelsa (AL). Twelve hogget Malpura rams, divided into four equal groups, were offered one of the four dietary treatments. A feeding-cum-metabolic trial was conducted to assess nutrient utilization. Rumen liquor samples were collected at 0, 3, 6, 12, 18 and 24 h post-feeding to assess rumen fermentation pattern and ciliate protozoa population. Inclusion of dried leaves in PMS-based diets improved CP and DCP content. Dietary DCP was low (P < 0.01) in PMS (8.52%) compared to tree leaves (9.77–11.59%) diets. AL and NL diets had higher (P < 0.05) DCP than the SL diet. The inclusion of tree leaves did not influence organic matter, crude protein or cellulose digestibility, but depressed dry matter, NDF, ADF and energy digestibility. DE content was also lower in tree leave diets. Inclusion of tree leaves improved CP and DCP intake, but DE intake and nitrogen utilization did not change. The pH of rumen liquor (SRL) was low (6.99, P < 0.05), but total nitrogen (52.9 mg/dl SRL) and NH₃-nitrogen (9.34 mg/dl SRL) concentrations were higher (P < 0.01) in the AL diet. TVFA concentrations and ciliate protozoa population were similar on the four diets. Animals in the four groups had the desired concentration of rumen metabolites required for fibrous diets. Complete feed-block feeding provided a constant nutrient supply to rumen microbes that optimise rumen fermentation. Blood biochemical constituents did not change due to the inclusion of tree leaves. Therefore, tree leaves can be included with roughage-based feeding to improve the protein nutrition status of ruminants. Further studies are required to assess the negative influence of tree leaves on digestibility.     

Effects of Yucca schidigera extract on fermentation and degradation of steroidal saponins in the rumen simulation technique (RUSITEC)

A rumen simulation technique (RUSITEC) apparatus with eight 940 ml fermentation vessels was used to study the effects of the steroidal saponins in Yucca schidigera extract (YE) on ruminal microbial activity and saponin degradation. The YE contained approximately 4.4% (w/w) saponin, as smilagenin equivalents, and was included at 0 (control) or 0.5 mg ml⁻¹ (n=4) in the McDougall's buffer infused continuously into the vessels (dilution rate=0.75 day⁻¹). Each vessel received 5 g chopped alfalfa hay and 5 g concentrate (as-fed basis) daily for 22 days. Ammonia concentrations were lower (P<0.05) in effluent from vessels receiving YE than from controls for the first half of the study, but did not differ thereafter. Total amounts of VFA in effluent were not affected (P>0.05) by YE, but molar proportions of iso-butyric and iso-valeric acids were lower (P<0.05) in the YE vessels than in the controls in the first half of the experiment. Yucca extract at 0.5 mg ml⁻¹ did not affect (P>0.05) dry matter disappearance (DMD) from hay or from concentrate, nor did it affect total gas or methane production, or bacterial numbers (total or cellulolytic populations) in homogenates prepared from fermenter vessel liquid and feed particles. Protozoal numbers in the homogenates were substantially reduced (P<0.01) by YE (at 0.5 mg ml⁻¹), protease activity was increased (P<0.05), deaminase activity and activity against Ala₂ were unaffected (P>0.05) and activity against Ala₅ was reduced by 25% (P>0.05). When the homogenates from control and YE-supplemented (0.5 mg ml⁻¹) vessels were used to inoculate roll tubes containing 0 or 5 mg ml⁻¹ of YE, fewer colonies developed (P<0.01) in roll tubes containing YE than in those without YE, irrespective of the source of inoculum. Homogenates were also assayed for saponin degradation and for protease, peptidase and deaminase activities. Inoculum from the vessels receiving YE degraded saponin slightly during a 2 h incubation. Yucca extract at 0.5 mg ml⁻¹ altered proteolytic activity and reduced protozoal numbers, but did not affect DMD or bacterial activity, and did not induce resistance to YE at a concentration of 5 mg ml⁻¹.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Physiological control of leaf methane emission from wetland plants

The transport of methane from the rhizosphere to the atmosphere takes place in the intercellular spaces and stomata of wetland plants, and foliar gas exchange is one of the critical steps of the transport process. The objectives of our research were to investigate: (i) variation in foliar gas exchange among four common wetland plant species (i.e., Peltandra virginica L., Orontium aquaticum L., Juncus effusus L., and Taxodium distichum L.), (ii) the role of key environmental factors (i.e., light, temperature, and carbon dioxide concentration) in controlling foliar methane emission, and (iii) physiological mechanisms underlying the variation in methane emission due to species and the environment. Experiments were conducted in an instantaneous, flow-through gas-exchange system that operated on a mass balance approach and concurrently measured foliar fluxes of methane, water vapor, and carbon dioxide. The chamber system allowed for the control of light, temperature, humidity, and carbon dioxide concentration. Diel patterns of methane emission varied among species, with daylight emissions from P. virginica and O. aquaticum 2–4 times those of J. effusus and T. distichum in saturating light. Foliar methane emission from P. virginica (1.80 μmol m⁻² s⁻¹) under ambient daylight conditions was an order of magnitude higher than that of the other three species (∼0.20 μmol m⁻² s⁻¹). As leaf temperature was increased by 10 °C, methane emission increased by a factor of 1.5–2.2, and the temperature effect was independent of stomatal conductance. When data were pooled among the four species, varying the light and carbon dioxide concentrations in a stepwise manner produced changes in foliar methane emission that were associated with stomatal conductance (r² = 0.52). To scale our observations to other wetland plant species, a stepwise multiple regression model is offered that incorporates stomatal conductance and net carbon dioxide assimilation to estimate instantaneous methane emission from foliar surfaces. The model indicates that changes in stomatal conductance affect methane emission three times more than equivalent changes in net carbon dioxide assimilation.     

Identification of microorganisms in the granules generated during methane fermentation of the syrup wastewater produced while canning fruit

The wastewater produced in the process of canning fruit contains a syrup that consists mainly of sucrose. This syrup wastewater was treated by methane fermentation in an upflow anaerobic sludge blanket reactor. The organic loading rate of syrup wastewater was increased gradually as fermentation progressed. The higher the organic loading rate, the more methane gas evolved until the organic loading rate reached 30.3 kg COD m^?3 d^?1, at which point methane generation abruptly diminished because the loading rate was too high to stably operate the reactor. The changes in the microbial community, that of both bacteria and archaea in the granules, were analyzed simultaneously using PCR-DGGE during the fermentation process. Methanosaeta spp., which are methanogenic archaea that produce extracellular polymers indispensable for the formation of granules, were dominant when the methane gas vigorously evolved, and the iron-reducing bacterium belonging to genus Geobacter, which outcompetes methanogens, grew proportionally with the deterioration of methane fermentation.