Short communication: Metabolic synthesis of vitamin D2 by the gut microbiome.

The origin of vitamin D2 in herbivorous animals was investigated in vivo in sheep and in bovine as well as mouse gastrointestinal tracts. A high concentration of 25-hydroxyvitamin D2 in blood plasma of sheep both in summer and winter appeared to be incompatible with the undetectable level of vitamin D2 in the pasture on which the sheep were grazing. Studies with bovine rumen contents from a cow grazing the same pasture as the sheep, demonstrated an increased concentration of vitamin D2 on anaerobic incubation in a 'Rusitec' artificial rumen, which was further enhanced when cellulose powder was added as a fermentation substrate. The colon contents of mice that were fed from weaning on a vitamin D-free diet were found to contain vitamin D2. The results of these comparative studies in 3 animal species indicated that vitamin D2 was being generated by microbial anaerobic metabolism in the gastrointestinal tract.

Feeding an unsalable carrot total-mixed ration altered bacterial amino acid degradation in the rumen of lambs.

The objective of this study was to determine the influence of a total-mixed ration including unsalable carrots at 45% DM on the rumen microbiome; and the plasma, rumen and liver metabolomes. Carrots discarded at processing were investigated as an energy-dense substitute for barley grain in a conventional feedlot diet, and improved feed conversion efficiency by 25%. Here, rumen fluid was collected from 34 Merino lambs at slaughter (n = 16 control; n = 18 carrot) after a feeding period of 11-weeks. The V4 region of the 16S rRNA gene was sequenced to profile archaeal and bacterial microbe communities. Further, a comprehensive, targeted profile of known metabolites was constructed for blood plasma, rumen fluid and biopsied liver metabolites using a gas chromatography mass spectrometry (GC-MS) metabolomics approach. An in vitro batch culture was used to characterise ruminal fermentation including gas and methane (CH4) production. In vivo rumen microbial community structure of carrot fed lambs was dissimilar (P < 0.01; PERMANOVA), and all measures of alpha diversity were greater (P < 0.01), compared to those fed the control diet. Unclassified genera in Bacteroidales (15.9 ± 6.74% relative abundance; RA) were more abundant (P < 0.01) in the rumen fluid of carrot-fed lambs, while unclassified taxa in the Succinivibrionaceae family (11.1 ± 3.85% RA) were greater (P < 0.01) in the control. The carrot diet improved in vitro ruminal fermentation evidenced as an 8% increase (P < 0.01) in DM digestibility and a 13.8% reduction (P = 0.01) in CH4 on a mg/ g DM basis, while the control diet increased (P = 0.04) percentage of propionate within total VFA by 20%. Fourteen rumen fluid metabolites and 27 liver metabolites were influenced (P ≤ 0.05) by diet, while no effect (P ≥ 0.05) was observed in plasma metabolites. The carrot diet enriched (impact value = 0.13; P = 0.01) the tyrosine metabolism pathway (acetoacetic acid, dopamine and pyruvate), while the control diet enriched (impact value = 0.42; P ≤ 0.02) starch and sucrose metabolism (trehalose and glucose) in rumen fluid. This study demonstrated that feeding 45% DM unsalable carrots diversified bacterial communities in the rumen. These dietary changes influenced pathways of tyrosine degradation, such that previous improvements in feed conversion efficiency in lambs could be explained.

A review on microalgae-mediated biotechnology for removing pharmaceutical contaminants in aqueous environments: Occurrence, fate, and removal mechanism.

Pharmaceutical compounds in aquatic environments have been considered as emerging contaminants due to their potential risks to living organisms. Microalgae-based technology showed the feasibility of removing pharmaceutical contaminants. This review summarizes the occurrence, classification, possible emission sources, and environmental risk of frequently detected pharmaceutical compounds in aqueous environments. The efficiency, mechanisms, and influencing factors for the removal of pharmaceutical compounds through microalgae-based technology are further discussed. Pharmaceutical compounds frequently detected in aqueous environments include antibiotics, hormones, analgesic and non-steroidal anti-inflammatory drugs (NSAIDs), cardiovascular agents, central nervous system drugs (CNS), antipsychotics, and antidepressants, with a concentration ranging from ng/L to µg/L. Microalgae-based technology majorly remove the pharmaceutical compounds through bioadsorption, bioaccumulation, biodegradation, photodegradation, and co-metabolism. This review identifies the opportunities and challenges for microalgae-based technology and proposed suggestions for future studies to tackle challenges. The findings of this review advance our understanding of the occurrence and fate of pharmaceutical contaminants in aqueous environments, highlighting the potential of microalgae-based technology for pharmaceutical contaminants removal.

Characterisation of the Faecal Microbiome of Foals from 0-5 Months of Age and Their Respective Mares across Five Geographic Locations.

BACKGROUND: A foal undergoes considerable growth and development from birth to weaning, progressing from a milk-based diet to complete herbivory. The symbiotic relationships between bacteria, archaea and fungi substantiate this energy demand by colonising the hindgut and remaining flexible throughout the diet transitions. METHODS: A total of 70 faecal samples were collected from 14 mares and their foals across five studs in NSW as they aged from 0 to 5 months old. DNA was extracted from faecal samples and underwent amplification and sequencing of the 16S rRNA gene V4 hypervariable region of archaea and bacteria, and the fungal internal transcribed spacer-1 (ITS1) region. The fungal and bacterial community structure was assessed using Bray-Curtis dissimilarities, and the effect of age at sampling and location was determined using PERMANOVA. RESULTS: Age at sampling had a substantial effect on the foal's archaeal and bacterial faecal microbiota (PERMANOVA: R2 = 0.16; p < 0.01), while the effect of geographical location was smaller but still significant (PERMANOVA: R2 = 0.07; p < 0.01). The overall abundance, diversity and richness of bacterial and archaeal populations increased (p < 0.01) as foals aged, most noticeably rising between foals 1 to 2 and 2 to 3 months of age. The 15 most relatively abundant fungal species were all environmental saprophytes, most strongly affected by geographical location (p < 0.01) rather than age at sampling. There was an effect of location on Preussia Africana (p = 0.02) and a location × age interaction for fungal species Preussia persica (p < 0.01), Acremonium furcatum (p = 0.04), and Podospora pseudocomata (p = 0.01). There was no effect of age, location, or location × age interaction on the relative abundance of the remaining fungal species. CONCLUSIONS: The faecal microbiome appeared to stabilise for most bacterial and archaeal genera by 2 to 3 months of age, resembling an adult mare. Bacterial genera isolated from faecal samples belonged mainly to the Firmicutes phylum. Age at sampling more strongly affected the archaeal and bacterial faecal microbiota than the effect of the geographical location where the horse was sampled. The lack of effect of location on microbe populations suggests that although environmental factors may influence population structure, there are distinct differences at each stage of foal maturation.

Ecotoxicological response of Spirulina platensis to coexisted copper and zinc in anaerobic digestion effluent.

Copper ion (Cu2+) and zinc ion (Zn2+) are widely co-existent in anaerobic digestion effluent as typical contaminants. This work aims to explore how Cu2+-Zn2+ association affects physiological properties of S. platensis using Schlösser medium (SM) and sterilized anaerobic digestion effluent (SADE). Microalgae cells viability, biochemical properties, uptake of Cu2+ and Zn2+, and risk assessment associated with the biomass reuse as additives to pigs were comprehensively assessed. Biomass production ranged from 0.03 to 0.28 g/L in SM and 0.63 to 0.79 g/L in SADE due to the presence of Cu2+ and Zn2+. Peak value of chlorophyll-a and carotenoid content during the experiment decreased by 70-100% and 40-100% in SM, and by 70-77% and 30-55% in SADE. Crude protein level reduced by 4-41% in SM and by 65-75% in SADE. The reduction ratio of these compounds was positively related to the Cu2+ and Zn2+ concentrations. Maximum value of saturated and unsaturated fatty acids was both obtained at 0.3 Cu + 2.0 Zn (50.8% and 22.8%, respectively) and 25% SADE reactors (33.8% and 27.7%, respectively). Uptake of Cu in biomass was facilitated by Zn2+ concentration (> 4.0 mg/L). Risk of S. platensis biomass associated with Cu2+ was higher than Zn2+. S. platensis from SM (Cu2+ ≤ 0.3 mg/L and Zn2+ ≤ 4.0 mg/L) and diluted SADE (25% and 50% SADE) reactors could be used as feed additives without any risk (hazard index <1), which provides sufficient protein and fatty acids for pig consumption. These results revealed the promising application of using S. platensis for bioremediation of Cu2+ and Zn2+ in anaerobic digestion effluent and harvesting biomass for animal feed additives.

Unsalable Vegetables Ensiled With Sorghum Promote Heterofermentative Lactic Acid Bacteria and Improve in vitro Rumen Fermentation.

This study characterized the nutritive and microbial profiles and the fermentation characteristics of silage with the following compositions on a dry matter (DM) basis: (1) 100% sorghum, (2) 70% sorghum + 30% carrot or pumpkin, and (3) 40% sorghum + 60% carrot or pumpkin. The treatments were further divided based on the addition or no addition of a probiotic inoculant. After 70 days of ensiling, the silage was incubated for 48 h using the in vitro batch culture technique. Crude protein and non-fiber carbohydrates in the silage increased (P ≤ 0.01) by 5.7 percent point (pp) and 9.6 pp, respectively, with pumpkin at 60% DM. The V4 region of the 16S rRNA gene was sequenced to profile pre-ensiled and ensiled archeal and bacterial communities. Silages containing carrot or pumpkin strongly influenced the microbial structure (PERMANOVA: R 2 = 0.75; P < 0.001), despite the ensiled treatments being dominated by Lactobacillus spp., except for the control, which was dominated by Weissella and Pediococcus spp. (P < 0.01). Linear discriminant analysis indicated that carrot and pumpkin silages were responsible for the increased relative abundance of Lactobacillus and Acinetobacter spp. (log LDA score ≥ 2), respectively. After 48 h of incubation, carrot and pumpkin inclusion increased (P < 0.01) the in vitro DM digestibility by 22.5 and 31.3%, increased the total volatile fatty acids (VFAs) by 16 and 20.6% (P < 0.01), respectively, and showed a tendency (P = 0.07) to increase the gas production. Therefore, this study supports the use of carrot or pumpkin in sorghum silages to maximize feed digestibility and total VFA concentrations.

Dose response of biochar and wood vinegar on in vitro batch culture ruminal fermentation using contrasting feed substrates.

Within Australia, approximately 6.4% of total greenhouse gas emissions are from animal methane (CH4) derived from enteric fermentation. Mitigation of ruminant CH4 is a key concept in support of sustainable agriculture production; dietary manipulations a viable strategy to lower CH4 release during enteric fermentation. In order to determine the effects of dose response of biochar and wood vinegar supplementation on fermentation parameters and CH4 production, this study utilized in vitro batch culture incubations. It is hypothesized that the addition of either biochar or wood vinegar will successfully reduce enteric CH4 emissions without negative modification of other fermentation parameters. Three feed substrates (vegetable mixed ration, maize silage, and winter pasture) were separated into treatments containing either biochar at 0%, 0.5%, 1%, 2%, and 4% DM replacing substrate (w/w basis), or wood vinegar at 0%, 0.25%, 0.5%, 1%, and 2% into incubation media volume (v/v). At 6, 12, and 24 hours after inoculation, total gas volume, and methane (CH4 %) were measured. Volatile fatty acid (VFA) concentrations, media pH, and in vitro dry matter digestibility were measured at 24 hours. Biochar at various dosages had no effect (P > 0.05) on fermentation characteristics other than decreased in vitro dry matter digestibility (IVDMD; P = 0.01) at 2% and 4% (DM basis) inclusion. Similar to biochar, dose response of wood vinegar had no effect on in vitro fermentation characteristics. However, feed substrate had major effects on all fermentation parameters (P = 0.01) where winter pasture > vegetable mixed ration > maize silage for all recorded fermentation characteristics. Biochar and wood vinegar supplementation were ineffectual in mitigating CH4 production or modifying fermentation characteristics, thus rejecting the initial hypothesis. These results suggest the use of biochar is not an effective tool for methane mitigation in ruminant livestock and infers that studies previously reporting success must better define the systemic mechanisms responsible for the reduction in CH4.

Ensiling sorghum with unsalable pumpkin improves feed digestibility with minimal influence on the rumen microbial population using the rumen simulation technique.

This study aimed to determine the influence of sorghum ensiled with unsalable pumpkin at 20 or 40% dry matter (DM) basis on rumen fermentation characteristics and rumen microbial communities using the rumen simulation technique (RUSITEC). The experiment used a completely randomised design including silages comprising (1) 100% sorghum; (2) 80% sorghum + 20% DM pumpkin; or (3) 60% sorghum + 40% DM pumpkin. Each RUSITEC run (n = 2) was 15 d long, including 6 d of adaptation and 9 d of sampling. Dry matter digestibility (DMD) was measured on d 8 and 10-13. Gas production was measured daily, whereas methane and volatile fatty acids (VFA) production were measured from d 7-15. Solid-associated microbes (SAM) were collected on d 5, 10 and 15, whereas liquid-associated microbes (LAM) were collected after 15-d incubation. The V4 region of the 16S rRNA gene and the ITS1 region were sequenced to identify archaeal, bacterial and fungal communities. Ensiling 40% DM pumpkin with sorghum increased DMD and decreased the ratio of acetate to propionate (P ≤ 0.01). Both bacterial SAM and LAM communities were dominated by Megasphaera, and had the highest relative abundance (P = 0.03) with 40% DM pumpkin after 5 d incubation in the SAM community, while species of the Aspergillus genus dominated fungal SAM and LAM communities with 20 or 40% DM unsalable pumpkin. Therefore, ensiling up to 40% DM unsalable pumpkin with sorghum produces a high-quality ruminant feed with minimal influence on the rumen microbial population. KEY POINTS: • Including 40% DM unsalable pumpkin decreased acetate:propionate • Ensiling unsalable pumpkin with sorghum increases digestibility in a RUSITEC • Rumen microbial communities were slightly influenced by unsalable pumpkin inclusion.

Feeding unsaleable carrots to lambs increased performance and carcass characteristics while maintaining meat quality.

This study investigated the effect of feeding unsaleable carrots to lambs within a total-mixed ration (TMR) on performance, carcass characteristics, meat quality and sensory parameters. Thirty-six Australian Merino wether lambs were fed a control (barley-based) or carrot-based TMR for 11-weeks. Carrot-fed lambs had 2.7% higher cold dressing percentage (P = 0.03) while consuming less than control lambs. Subcutaneous fat of carrot-fed lambs contained less branch-chained, and more cis- and trans-monounsaturated fatty acids (FA; P ≤ 0.01) compared to control-fed lambs, which tended (P = 0.08) to have higher concentrations of polyunsaturated FA, despite the Longissimus lumborum (LL) muscle being unchanged by diet. Under retail display conditions, L* and hue values were lower (P ≤ 0.04) for 5 d aged LL samples from carrot-fed lambs. No differences were observed in other meat quality and sensory parameters between diets. Therefore, feeding unsaleable carrots at 45% DM in a TMR can improve lamb performance and carcass characteristics, while maintaining meat quality and FA composition.

Ozone Decreased Enteric Methane Production by 20% in an in vitro Rumen Fermentation System.

Ozone (O3) is volatile, highly oxidative, and has theoretical potential to reduce ruminant enteric methanogenesis by interactions between archaea and bacteria, and substrate and oxygen. The effects of O3 on the rumen microbiota, fermentation parameters, and CH4 emissions were studied through in vitro fermentation using a RUSITEC apparatus with O3 dissolved in the salivary buffer. The substrate consisted of maize silage or grain concentrates, and the treatments were (1) control (no O3) and (2) O3 at 0.07 ± 0.022 mg/L in the buffer. A 4-day adaptation period followed by a 6-day experimental period was used for measuring gas production and composition, as well as fermentation characteristics, which included ruminal volatile fatty acids (VFA) and liquid- and solid-associated microbial communities. Ozone treatment decreased total gas production by 15.4%, most notably CH4 production by 20.4%, and CH4 gas concentration by 5.8%, without compromising dry matter digestibility (DMD) of either maize silage or grain concentrates. There were no significant effects of O3 treatment on VFA production or pH. Ozone treatment reduced the relative abundance of methanogens, particularly Methanomicrobium. This study demonstrates the potential use of O3 as a method to reduce ruminant enteric methanogenesis.

Influence of probiotics on biofilm formation and diversity of bacteria colonising crop sorghum ensiled with unsalable vegetables.

The objective of this study was to characterise in situ digestion kinetics and bacterial colonisation of crop sorghum ensiled with unsalable carrot or pumpkin at 0, 20 or 40% dry matter (DM). Silages with or without the application of a commercial probiotic were incubated in situ for 0, 3, 6, 9, 24 and 48 h. Calculation of in situ digestion kinetics was conducted for DM, organic matter and neutral detergent fibre (aNDF). The V4 region of the 16S rRNA gene was sequenced to determine the composition and diversity of bacteria colonising the silage. Organic matter and DM digestion kinetics indicated that greater vegetable inclusion increased (P < 0.05) the soluble fraction and effective degradability. Bacterial richness at 48 h incubation was greater (P = 0.02) in 20% carrot and 40% pumpkin treatments, compared with the control. An effect of level × probiotic was observed with increased Shannon diversity (P = 0.01) for 40% carrot and 20% pumpkin probiotic treatments, respectively. Primary colonising bacteria were members of the Prevotella genus, dominating after 3 and 6 h of incubation. The abundance of Prevotella increased by 4.1% at 3 h (P < 0.01) and by 4.7% at 9 h incubation with probiotics, compared with the control. Secondary biofilm colonisers included members of Treponema, Saccharofermentans, Fibrobacter, Ruminobacter and Anaerosporobacter genera, dominant from 9 h incubation onward. This study demonstrated that including unsalable vegetables at 20 or 40% DM increases the soluble fraction and effective degradability of sorghum silage during in situ digestion and increases diversity of bacteria colonising ensiled vegetables within the rumen. KEY POINTS: • Ensiling unsalable vegetables is a viable strategy to reduce food waste. • Ensiled vegetables increased in situ soluble fraction and effective degradability. • Bacterial richness at 48 h incubation improved with 20% carrot or 40% pumpkin. • Diversity of colonising rumen bacteria increased with carrot or pumpkin inclusion.

Effect of a pine enhanced biochar on growth performance, carcass quality, and feeding behavior of feedlot steers.

The objective of this study was to evaluate the effect of enhanced biochar (EB) on growth performance, carcass quality, and feeding behavior of feedlot steers fed high-forage and high-grain diets. A total of 160 crossbred steers (initial 286 ± 26 kg body weight [BW]) were blocked by BW and randomly assigned to 16 pens (10 steers per pen), 8 of which were equipped with the GrowSafe system for monitoring feeding behavior. Treatments were EB included in the diet at 0% (control), 0.5%, 1.0%, or 2.0% (dry matter [DM] basis) with four pens per treatment. The backgrounding phase (84 d) was divided into four 21-d periods, and the finishing phase (112 d) was divided into four 28-d periods, with a 28-d transition period for dietary adaptation. Pen was the experimental unit for all parameters except for feeding behavior, where steer was considered the experimental unit. Treatment was included as a fixed effect, and period was considered a repeated measure. Total weight gain and overall average daily gain (ADG) tended to decrease (P = 0.06) with 2.0% EB. There was no effect (P ≥ 0.13) of EB on dry matter intake (DMI), gain-to-feed ratio (G:F), net energy for gain, ADG, or final BW for the backgrounding or finishing phases. There was a treatment × period effect (P < 0.05) of EB on DMI, ADG, and G:F for both backgrounding and finishing phases. Hot carcass weight, dressing %, back fat, rib-eye area, and meat yield were not affected (P ≥ 0.26) by EB. Lean meat yield was increased (P = 0.03) by 2.0% EB compared to all other treatments. Compared to the control, 2.0% EB increased (P = 0.02) the number of carcasses that achieved Canada 1 grade. More (P = 0.05) carcasses from control steers were graded as Canada 3 as compared to those fed 0.5% or 2.0% EB. Quality grade and incidences of liver abscesses were not affected (P ≥ 0.44) by EB. Enhanced biochar had no effect (P ≥ 0.11) on feeding behavior during backgrounding or finishing phases. In conclusion, EB did not result in changes in growth rate, feed efficiency, or feeding behavior in feedlot cattle, but 2.0% EB increased lean carcass yield grade.

Derivation of volatile fatty acid from crop residues digestion using a rumen membrane bioreactor: A feasibility study.

This study evaluates the feasibility of a novel rumen membrane bioreactor (rumen MBR) to produce volatile fatty acids (VFA) from crop residues (i.e. lignocellulosic biomass). Rumen MBR can provide a sustainable route for VFA production by mimicking the digestive system of ruminant animals. Rumen fluid was inoculated in a reactor coupled with ultrafiltration (UF) membrane and fed with maize silage and concentrate feed at 60:40% (w/w). Continuous VFA production was achieved at an average daily yield of 438 mg VFA/g substrate. The most abundant VFA were acetic (40-80%) and propionic (10-40%) acids. The majority (73 ± 15%) of produced VFA was transferred through the UF membrane. Shifts in dominant rumen microbes were observed upon the transition from in vivo to in vitro environment and during reactor operation, however, stable VFA yield was maintained for 35 days, providing the first proof-of-concept of a viable rumen MBR.

Effects of inclusion of purple prairie clover (Dalea purpurea Vent.) with native cool-season grasses on in vitro fermentation and in situ digestibility of mixed forages.

BACKGROUND: Incorporation of legume species into native North American pastures is considered an effective method to increase native pasture productivity and improve the nutritive value of forage. This study evaluated the effects of inclusion of purple prairie clover (PPC, Dalea purpurea Vent.), a native legume forage, with native cool-season grasses on the in vitro fermentation and in situ digestibility of mixed forages. METHODS: Whole plant PPC and mixtures of cool-season grasses were harvested when the PPC reached the vegetative (VEG), full flower (FL) and seedpod (SP) stages, and were combined in ratios (DM basis) of 0:100, 25:75, 50:50, 75:25 and 100:0 at each maturity. In vitro ruminal incubations using these mixtures were conducted for 48 h to determine gas production (GP), in vitro DM disappearance (IVDMD), total volatile fatty acids (VFA) and ammonia-N production. Mixtures of forages harvested when the PPC reached the FL stage and 50:50 mixture of forages harvested at VEG, FL and SP stages were incubated in the rumen of three heifers for 0, 2, 6, 12, 24, 48, 72 and 96 h to determine in situ degradabilities of DM, neutral detergent fibre (aNDF) and crude protein (CP). RESULTS: Contents of aNDF and ADF increased (P < 0.01), while CP decreased (P < 0.001) as PPC matured. Concentrations of extractable condensed tannins in PPC ranked as FL > VEG > SP (P < 0.05). Regardless of PPC proportions in the mixture, GP decreased (P < 0.05) with increasing PPC maturity. Increasing PPC proportions linearly increased (P < 0.001) GP, IVDMD and total VFA at VEG, but linearly decreased (P < 0.001) them at SP. Irrespective of PPC maturity, ammonia-N production linearly increased (P < 0.01) with increasing proportions of PPC and the concentration was higher (P < 0.05) at VEG than at FL and SP stages. Increasing proportion of PPC at either maturity linearly increased (P < 0.001) molar percentage of acetate (A) and branched-chain VFA, but linearly decreased (P < 0.001) molar percentage of propionate (P), resulting in a linearly increase (P < 0.001) in the A:P ratio. Increasing FL PPC in the mixture linearly and quadratically (P < 0.01) increased a (soluble fraction), but linearly and quadratically decreased (P < 0.01) b (potentially degradable fraction) for DM and aNDF, resulting in linear (P < 0.05) and quadratic (P < 0.01) increases in DM and aNDF maximum potential degradabilities (a + b). Effective degradabilities of DM and aNDF were also linearly and quadratically increased (P < 0.05), and CP was quadratically increased (P < 0.05) with increasing FL PPC, with the greatest effective degradability being observed with ratios between 50:50 and 75:25. Ruminal maximum potential degradabilities of DM and aNDF decreased (P < 0.001) as the forage matured. Effective degradability of DM ranked as VEG > FL > SP (P < 0.001), whereas the effective degradability of aNDF was similar between VEG and FL and both were greater (P < 0.01) than SP. CONCLUSIONS: Inclusion of vegetative PPC in a mixed forage diet resulted in the greatest digestibility and incorporation of PPC before seedpod stage with native grasses had a positive effect on ruminal fermentation. Effects of PPC on ruminal digestion depend on both the stage of maturity and its proportion in mixed legume-grass pastures. Pastures containing 50% of PPC in full flower stage would likely provide the greatest quality diet to grazing ruminants subject to potential animal selectivity.

Crop Sorghum Ensiled With Unsalable Vegetables Increases Silage Microbial Diversity.

Ensiling vegetables with forage crops is a suggested method of waste diversion and can be directly utilized as a livestock feed. Carrot or pumpkin, ensiled at 0, 20, or 40% dry matter (DM) with crop sorghum, and with or without a second-generation silage inoculant were assessed for nutritive composition, organic acid profiles, aerobic stability and in vitro rumen fermentation characteristics. The study was a completely randomized design, with the fixed effects consisting of vegetable type (carrot vs. pumpkin), level (i.e., the level of vegetables), inoculant (inoculant or non-inoculant) and the interactions, and mini-silos within treatment as the random effect. The experimental unit for sorghum treatments represented by each mini-silo (5 kg capacity). Silage was sampled after 70-days ensiling for nutrient composition, 14-day aerobic stability, organic acid profiles and microbial diversity. After 24 h in vitro incubation, rumen fermentation parameters were assessed, measuring gas and methane (CH4) production, in vitro digestibility and volatile fatty acid concentrations. Sorghum ensiled with carrot or pumpkin at 20% or 40% DM increased crude fat (P ≤ 0.01) and decreased (P ≤ 0.01) silage surface temperature upon aerobic exposure compared to the control. Bacterial communities analyzed through 16S rRNA gene sequencing linearly increased (P ≤ 0.01) in diversity across both vegetables when the vegetable proportion was increased in the silage; dominated by Lactobacillus species. ITS analysis of the fungal microbiota upon silage opening and after 14 days (aerobic stability) identified increased (P ≤ 0.03) fungal diversity with increasing vegetable proportions, predominantly populated by Fusarium denticulatum, Issatchenkia orientalis, Kazachstania humilis, and Monascus purpureus. Upon assessment in vitro, there was an increase (P ≤ 0.04) in in vitro digestibility and some CH4 parameters (% CH4, and mg CH4/g DM), with no effect (P ≥ 0.17) on remaining CH4 parameters (mL CH4/g DM, mg CH4/g digested DM), gas production or pH. However, increasing vegetable amount decreased percentage of acetic acid and increased percentage of propionic acid of the total VFA, decreasing A:P ratio and total VFA concentration as a result (P ≤ 0.01). The results from this study indicate including carrot or pumpkin at 20 or 40% DM in a sorghum silage can produce a highly digestible, microbially diverse and energy-rich livestock feed.

A Pine Enhanced Biochar Does Not Decrease Enteric CH4 Emissions, but Alters the Rumen Microbiota.

The objective of this study was to examine the effect of a pine enhanced biochar (EB) on rumen fermentation, apparent total tract digestibility, methane (CH4) emissions, and the rumen and fecal microbiome of Angus × Hereford heifers fed a barley silage-based diet. The experiment was a replicated 4 × 4 Latin square using 8 ruminally cannulated heifers (565 ± 35 kg initial BW). The basal diet contained 60% barley silage, 35% barley grain and 5% mineral supplement with EB added at 0% (control), 0.5, 1.0, or 2.0% (DM basis). Each period lasted 28 days, consisting of 14 days adaptation and 14 days of measurements. Samples for profiling of the microbiome in rumen liquid, solids and feces were collected on d 15 before feeding. Rumen samples for fermentation characterization were taken at 0, 3, 6, and 12 h post feeding. Total collection of urine and feces was conducted from days 18 to 22. Heifers were housed in open-circuit respiratory chambers on days 26-28 to estimate CH4 emissions. Ruminal pH was recorded at 1-min intervals during CH4 measurements using indwelling pH loggers. Data were analyzed with the fixed effects of dietary treatment and random effects of square, heifer within square and period. Dry matter intake was similar across treatments (P = 0.21). Ammonia N concentration and protozoa counts responded quadratically (P = 0.01) to EB in which both were decreased by EB included at 0.5 and 1.0%, compared to the control and 2.0% EB. Minimum pH was increased (P = 0.04), and variation of pH was decreased (P = 0.03) by 2.0% EB. Total tract digestibility, N balance and CH4 production were not affected (P ≥ 0.17) by EB. Enhanced biochar decreased the relative abundance of Fibrobacter (P = 0.05) and Tenericutes (P = 0.01), and increased the relative abundance of Spirochaetaes (P = 0.01), Verrucomicrobia (P = 0.02), and Elusimicrobia (P = 0.02). Results suggest that at the examined concentrations, EB was ineffective at decreasing enteric CH4 emissions, but did alter specific rumen microbiota.

Microbial characterization and fermentative characteristics of crop maize ensiled with unsalable vegetables.

Incorporation of carrot or pumpkin at 0, 20 or 40% dry matter (DM-basis) with crop maize, with or without a silage inoculant was evaluated after 70 days ensiling for microbial community diversity, nutrient composition, and aerobic stability. Inclusion of carrots or pumpkin had a strong effect on the silage bacterial community structure but not the fungal community. Bacterial microbial richness was also reduced (P = 0.01) by increasing vegetable proportion. Inverse Simpson's diversity increased (P = 0.04) by 18.3% with carrot maize silage as opposed to pumpkin maize silage at 20 or 40% DM. After 70 d ensiling, silage bacterial microbiota was dominated by Lactobacillus spp. and the fungal microbiota by Candida tropicalis, Kazachstania humilis and Fusarium denticulatum. After 14 d aerobic exposure, fungal diversity was not influenced (P ≥ 0.13) by vegetable type or proportion of inclusion in the silage. Inoculation of vegetable silage lowered silage surface temperatures on day-7 (P = 0.03) and day-14 (P ≤ 0.01) of aerobic stability analysis. Our findings suggest that ensiling unsalable vegetables with crop maize can successfully replace forage at 20 or 40% DM to produce a high-quality livestock feed.

Effects of Hardwood Biochar on Methane Production, Fermentation Characteristics, and the Rumen Microbiota Using Rumen Simulation.

Biochar is a novel carbonized feed additive sourced from pyrolyzed biomass. This compound is known to adsorb gasses and carbon, participate in biological redox reactions and provide habitat biofilms for desirable microbiota proliferation. Therefore, biochar holds potential to modify rumen fermentation characteristics and reduce enteric CH4 emissions. The objective of this study was to investigate the effect of hardwood biochar supplementation on fermentation parameters, methane (CH4) production and the ruminal archaeal, bacterial, and fungal microbiota using the in vitro RUSITEC (rumen simulation technique) system. Treatments consisted of a control diet (oaten pasture: maize silage: concentrate, 35:35:30 w/w) and hardwood biochar included at 400 or 800 mg per day (3.6 and 7.2% of substrate DM, respectively), over a 15-day period. Biochar supplementation had no effect (P ≥ 0.37) on pH, effluent (mL/d), total gas (mL/d), dry matter (DM) digestibility or CH4 production (mg/d). The addition of 800 mg biochar per day had the tendency (P = 0.10) to lower the % of CH4 released in fermentation compared to 400 mg/d biochar treatment. However, no effect (P ≥ 0.44) was seen on total VFA, acetate, propionate, butyric, branched-chain VFA, valerate and caproate production and the ratio of acetate to propionate. No effect (P > 0.05) was observed on bacterial, archaeal or fungal community structure. However, biochar supplementation at 800 mg/d decreased the abundance of one Methanomethylophilaceae OTU (19.8-fold, P = 0.046) and one Lactobacillus spp. OTU (31.7-fold, P < 0.01), in comparison to control treatments. Two fungal OTUs classified as Vishniacozyma victoriae (5.4 × 107 increase) and Sporobolomyces ruberrimus (5.4 × 107-fold increase) were more abundant in the 800 mg/d biochar samples. In conclusion, hardwood biochar had no effects on ruminal fermentation characteristics and may potentially lower the concentration of enteric CH4 when included at higher dosages by manipulating ruminal microbiota abundances.

Application of rumen and anaerobic sludge microbes for bio harvesting from lignocellulosic biomass.

This study investigated the production of biogas, volatile fatty acids (VFAs), and other soluble organic from lignocellulosic biomass by two microbial communities (i.e. rumen fluid and anaerobic sludge). Four types of abundant lignocellulosic biomass (i.e. wheat straw, oaten hay, lurence hay and corn silage) found in Australia were used. The results show that rumen microbes produced four-time higher VFAs level than that of anaerobic sludge reactors, indicating the possible application of rumen microorganism for VFAs generation from lignocellulosic biomass. VFA production in the rumen fluid reactors was probably due to the presence of specific hydrolytic and acidogenic bacteria (e.g. Fibrobacter and Prevotella). VFA production corroborated from the observation of pH drop in the rumen fluid reactors indicated hydrolytic and acidogenic inhibition, suggesting the continuous extraction of VFAs from the reactor. Anaerobic sludge reactors on the other hand, produced more biogas than that of rumen fluid reactors. This observation was consistent with the abundance of methanogens in anaerobic sludge inoculum (3.98% of total microbes) compared to rumen fluid (0.11%). VFA production from lignocellulosic biomass is the building block chemical for bioplastic, biohydrogen and biofuel. The results from this study provide important foundation for the development of engineered systems to generate VFAs from lignocellulosic biomass.

Effect of humic substances on rumen fermentation, nutrient digestibility, methane emissions, and rumen microbiota in beef heifers1.

Ruminants play an important role in food security, but there is a growing concern about the impact of cattle on the environment, particularly regarding greenhouse gas emissions. The objective of this study was to examine the effect of humic substances (HS) on rumen fermentation, nutrient digestibility, methane (CH4) emissions, and the rumen microbiome of beef heifers fed a barley silage-based diet. The experiment was designed as a replicated 4 × 4 Latin square using 8 ruminally cannulated Angus × Hereford heifers (758 ± 40.7 kg initial BW). Heifers were offered a basal diet consisting of 60% barley silage and 40% concentrate (DM basis) with either 0- (control), 100-, 200- or 300-mg granulated HS/kg BW. Each period was 28 d with 14 d of adaptation. Rumen samples were taken on day 15 at 0, 3, 6, and 12 h postfeeding. Total urine and feces were collected from days 18 to 22. Blood samples were taken on day 22 at 0 and 6 h postfeeding. Between days 26 and 28, heifers were placed in open-circuit respiratory chambers to measure CH4. Ruminal pH was recorded continuously during the periods of CH4 measurement using indwelling pH loggers. Intake was similar (P = 0.47) across treatments. Concentration of ammonia-N and counts of rumen protozoa responded quadratically (P = 0.03), where both increased at H100 and then decreased for the H300 treatments. Apparent total tract digestibility of CP (P = 0.04) was linearly increased by HS and total N retention (g/d, % N intake, g/kg BW0.75) was improved (P = 0.04) for HS when compared with the control. There was no effect of HS on CH4 production (g/d; P = 0.83); however, HS decreased the relative abundance of Proteobacteria (P = 0.04) and increased the relative abundance of Synergistetes (P = 0.01) and Euryarchaeota (P = 0.04). Results suggest that HS included at up to 300 mg/kg BW may improve N retention and CP digestibility, but there was no impact on CH4 production.