Substrate Dry Matter Research Articles

Utilization of citrus, date, and jujube substrates for anaerobic digestion processes

AbstractThis research explores the potential for generating biogas and clean energy by processing organic waste, a process that can become a sustainable solution to Syria's energy needs. Focusing on agricultural residues generated from citrus fruit orange, date, and jujube cultivation in Syria, this study evaluates the potential for anaerobic digestion of these residues for biogas production. It highlights the influence of substrate composition and the optimization of fermentation processes on biogas and methane production. The study focuses on evaluating the anaerobic digestion process by examining various dosages ranging from 20% to 50% dry matter for citrus orange waste, and different types of substrate with a fixed ratio of 20% substrate dry matter. It specifically discusses the factors influencing the inhibitory effect of anaerobic digestion, giving particular consideration to orange waste, a significant byproduct of the citrus industry. The biogas produced maintained a stable methane content when a citrus‐to‐inoculum ratio of 30:70 was used. Jujube waste, characterized by a composition rich in cellulose and hemicellulose, exhibited a higher potential for biogas and methane generation among the fruit waste investigated, particularly when combined with the inoculum in a 20:80 ratio. The research findings underscore the potential of using Syrian agricultural residues, including orange citrus peel, date, and jujube fruit, for the production of biogas through anaerobic digestion.

87 Evaluating the effects of an amino acid-formulated diet on ruminal fermentation in vitro

Abstract The objective was to determine the effect of an amino acid-formulated diet on ruminal fermentation in vitro. While most amino acid-formulated diets focus on improving the amino acid composition of rumen undegradable protein, additional benefits to microbial fermentation within the rumen are unknown. The experiment was a completely randomized design with a factorial treatment arrangement (2 donor diets and 2 substrates). Four ruminally cannulated steers were fed one of two donor diets: control (CON; 55% dry rolled corn, 20% corn silage, 15% dry distiller’s grain, and 10% supplement) and amino acid-formulated (AAF; 51% dry rolled corn, 15% corn silage, 14% dry distillers grain, 10% heat-treated soybean meal, and 10% supplement). After a 30-d diet adaptation period, rumen fluid was collected and pooled by donor diet for the in vitro fermentation. Additionally, two different substrates were used to represent each donor diet. Flasks contained 2.5 g dry matter (DM) of substrate and 150 mL of rumen fluid mixture (2 McDougall’s buffer:1 rumen fluid). Treatments were evaluated in triplicate flasks in two replicated runs for a total of six observations per treatment. Four sealed Ankom F57 bags were filled with 0.25 g DM of substrate each to facilitate measuring in vitro DM disappearance (IVDMD). Flasks were incubated at 39°C with sample aliquots collected at h 0, 4, 8, 12, and 24 to determine pH, ammonia, and volatile fatty acids (VFA). Data were analyzed using the MIXED procedure of SAS 9.4 with a repeated measures analysis. Ammonia, pH, and VFA were analyzed with effects of donor diet, substrate, time, and all interactions. A treatment by hour interaction was detected (P < 0.01) for pH; fluid from steers fed CON was greater than AAF at h 4, 8, 12, and 24. A treatment by hour interaction was also detected (P = 0.02) for ammonia; fluid from steers fed CON was greater than AAF at h 24, but was not different at other time points. Total VFA was affected by hour (P < 0.01) as it increased over time, but substrate did not have an effect (P = 0.21). A treatment by hour interaction (P = 0.06) tended to be observed for molar proportions of acetate as AAF and CON were similar at h 0 but AAF was increasing greater than CON over time. A treatment by hour interaction was observed (P = 0.01) for molar proportions of propionate; at h 4 at 8, CON was greater than AAF. No effect (P ≥ 0.32) of donor diet, substrate or their interaction were observed on IVDMD. Overall, an amino acid-formulated diet for cannulated steers did impact ruminal fermentation in vitro, mostly altering molar proportions of volatile fatty acids and ammonia release from protein degradation.

Effects of yeast culture on in vitro ruminal fermentation and microbial community of high concentrate diet in sheep.

This research aimed to investigate effects of different yeast culture (YC) levels on in vitro fermentation characteristics and bacterial and fungal community under high concentrate diet. A total of 5 groups were included in the experiment: control group without YC (CON), YC1 (0.5% YC proportion of substrate dry matter), YC2 (1%), YC3 (1.5%) and YC4 (2%). After 48h of fermentation, the incubation fluids and residues were collected to analyze the ruminal fermentation parameters and bacterial and fungal community. Results showed that the ruminal fluid pH of YC2 and YC4 groups was higher (P < 0.05) than that of CON group. Compared with CON group, the microbial protein, propionate and butyrate concentrations and cumulative gas production at 48h of YC2 group were significantly increased (P < 0.05), whereas an opposite trend of ammonia nitrogen and lactate was observed between two groups. Microbial analysis showed that the Chao1 and Shannon indexes of YC2 group were higher (P < 0.05) than those of CON group. Additionally, YC supplementation significantly decreased (P < 0.05) Succinivibrionaceae_UCG-001, Streptococcus bovis and Neosetophoma relative abundances. An opposite tendency of Aspergillus abundance was found between CON and YC treatments. Compared with CON group, the relative abundances of Prevotella, Succiniclasticum, Butyrivibrio and Megasphaera elsdenii were significantly increased (P < 0.05) in YC2 group, while Apiotrichum and unclassified Clostridiales relative abundances were decreased (P < 0.05). In conclusion, high concentrate substrate supplemented with appropriate YC (1%) can improve ruminal fermentation and regulate bacterial and fungal composition.

PSI-A-4 The Effects of a Novel Brewer’s Yeast Product on Ruminal Fiber Fermentation in Vitro

Abstract The objective was to evaluate a novel brewer’s yeast product on ruminal fiber fermentation in vitro. The experiment was a completely randomized design and included 4 treatments. Treatments included a negative control (NCON), a commercial brewer’s yeast product (Emprove MX; EMX), and a prototype concentrated brewer’s yeast (T1R8) fed at low and high inclusions (T1R8-L and T1R8-H, respectively). Inclusions for low and high treatments were 0.0008 and 0.0015 g, respectively, to correspond with 7 and 14 g inclusion in a lactating cow diet. Treatments were evaluated in duplicate flasks in 2 replicated runs for a total of four observations per treatment. Flasks contained 2.5 grams dry matter (DM) of substrate to represent a lactating cow diet (30% haylage, 25% corn silage, 20% corn, 15% canola meal, and 10% soybean hulls), and 150mL of rumen fluid mixture (2 McDougall’s buffer:1 rumen fluid ratio). Four F57 sealed Ankom bags were filled with 0.25 grams DM of substrate each to facilitate measuring in vitro DM and neutral detergent fiber disappearance (IVDMD and IVNDFD, respectively). Flasks were incubated at 39°C for 30 hours with sample aliquots collected at hour 0, 8, 16, and 30 to determine pH and volatile fatty acids (VFA). Data were analyzed using the MIXED procedure of SAS 9.4 with IVDMD and IVNDFD considered a main effect of treatment while pH and VFA were analyzed with effects of treatment, time, and their interaction. No differences in IVDMD were observed (P = 0.31) among treatments. Treatment affected (P = 0.01) IVNDFD with EMX (50.6%), T1R8-L (48.6%), and T1R8-H (50.3%) being greater than NCON (45.7%). A treatment by hour interaction (P &amp;lt; 0.01) was observed for total VFA concentration; EMX (71.8 mM), T1R8-L (72.6 mM), and T1R8-H (66.0 mM) were greater than NCON (47.8 mM) at h 8, but all treatments were not different at h 16. A treatment by hour interaction (P &amp;lt; 0.01) was also observed for acetate and propionate; EMX, T1R8-H, and T1R8-L were greater than NCON at h 8, but all treatments were not different at h 16. At h 30, greater propionate was observed for T1R8-H compared with all treatments. No effects of treatment or treatment by hour (P ≥ 0.89) were observed for acetate:propionate ratio. Although a treatment by hour interaction was not observed for pH (P = 0.12), a treatment effect (P = 0.03) indicated T1R8-L was the greatest with EMX being intermediate, and NCON and T1R8-H being the least. Novel brewer’s yeast product T1R8 increased the rate of substrate fermentation in the initial 8 h and increased the extent of fiber degradation after a 30 h in vitro fermentation.

Oil-in-Water Nanoemulsion Can Modulate the Fermentation, Fatty Acid Accumulation, and the Microbial Population in Rumen Batch Cultures.

In this study, three oil-in-water nanoemulsions were tested in two stages: In the first stage, three levels (on the substrate dry matter (DM)), namely 3%, 6%, and 9%, of three different oils, olive oil (OO), corn oil (CO), and linseed oil (LO), in raw and nanoemulsified (N) forms were used separately in three consecutive rumen batch cultures trials. The second stage, which was based on the first stage's results, consisted of a batch culture trial that compared the raw and nanoemulsified (N) forms of all three oils together, provided at 3% of the DM. In the first stage, NOO, NCO, and NLO preserved higher unsaturated fatty acid (UFA) and less saturated fatty acid (SFA) compared to OO, CO, and LO, respectively; noticeably, NCO had UFA:SFA = 1.01, 1.16, and 1.34 compared to CO, which had UFA:SFA = 0.66, 0.69, and 0.72 when supplemented at 3%, 6%, 9% of DM, respectively. In the second stage, UFA:SFA = 1.04, 1.12, and 1.07 for NOO, NCO, NLO, as compared to UFA:SFA = 0.69, 0.68, and 0.72 for OO, CO, and LO supplemented at 3% of DM. In conclusion, oil-in-water nanoemulsions showed an ability to decrease the transformation of UFA to SFA in the biohydrogenation environment without affecting the rumen microorganisms.

Phytonutrients in Red Amaranth (Amaranthus cruentus, L.) and Feed Ratios Enhanced Rumen Fermentation Dynamics, Suppress Protozoal Population, and Methane Production

The search for alternative modifiers of rumen fermentation to improve the production efficiency of livestock production is highly essential. Thisin vitrofermentation experiment was conducted using a factorial arrangement of two ratios of roughage to concentrate and seven levels of red amaranth (Amaranthus cruentusL.) leaf powder (RALP) as a percentage of total substrate in a completely randomized design (CRD). There were two factors: factor A was two ratios of roughage (R) to concentrate (C) at 60:40 and 40:60 and factor B was levels of RALP supplementation at 0, 2, 4, 6, 8, 10, and 12% dry matter (DM) of total dietary substrate. The results revealed that a R:C ratio at 40:60 increased rumen fermentation and reduced methane production (p&amp;lt; 0.05). The RALP incorporation as a feed additive was highly promising in enhancing propionate (C3) concentration, reducing acetate (C2) to (C3) ratio, and the protozoal population, while mitigating methane (CH4) production. Furthermore, DM degradation percentages were remarkably enhanced by increasing the RALP levels and R:C ratio at 40:60. In conclusion, plants rich in phytonutrients and minerals such as RALP and the lower R:C ratio showed a promising role in modulating rumen fermentation, mitigating methane production, as well as increasing substrate DM degradability.

Obtaining Cellulose-Available Raw Materials by Pretreatment of Common Agro-Forestry Residues With Pleurotus spp.

The goals of the present study were to characterize the profile of ligninolytic enzymes in five Pleurotus species and determine their ability to delignify eight common agro-forestry residues. Generally, corn stalks were the optimal inducer of Mn-dependent peroxidase activity, but the activity peak was noted after wheat straw fermentation by P. eryngii (3066.92 U/L). P. florida was the best producer of versatile peroxidase, especially on wheat straw (3028.41 U/L), while apple sawdust induced the highest level of laccase activity in P. ostreatus (49601.82 U/L). Efficiency of the studied enzymes was expressed in terms of substrate dry matter loss, which was more substrate-than species-dependent. Reduction of substrate dry mass ranged between 24.83% in wheat straw and 8.83% in plum sawdust as a result of fermentation with P. florida and P. pulmonarius, respectively. The extent of delignification of the studied substrates was different, ranging from 51.97% after wheat straw fermentation by P. pulmonarius to 4.18% in grapevine sawdust fermented by P. ostreatus. P. pulmonarius was also characterized by the highest cellulose enrichment (6.54) and P. ostreatus by very low one (1.55). The tested biomass is a highly abundant but underutilized source of numerous value-added products, and a cocktail of ligninolytic enzymes of Pleurotus spp. could be useful for its environmentally and economically friendly transformation.

Sulfur, fresh cassava root and urea independently enhanced gas production, ruminal characteristics and in vitro degradability

BackgroundTotal fresh cassava root (FCR) production was 275 million tonnes in 2018 which equals 61.1 % of the total production, and Thailand produced 10.7 % FCR of the total production. FCR is one of the main energy source for ruminant. The limitation of FCR utilization is due to the presence of hydrogen cyanide (HCN). The study aimed to evaluate the effect of sulfur, urea and FCR at various levels on in vitro gas production, ruminal fermentation and in vitro degradability. The study hypothesized that: (1) sulfur, urea and FCR have no interaction effect and (2) effect of FCR and urea is related to sulfur addition.ResultsThe study aimed to elucidate the optimum level of elemental sulfur, fresh cassava root (FCR) and urea and their effect on in vitro gas production, ruminal fermentation, thiocyanate concentration, and in vitro degradability. A 3 × 2 × 4 in a completely randomized design were conducted. Factor A was level of sulfur at 0 %, 1 and 2 % of concentrate dry matter (DM), factor B was level of urea at 2 and 4 % of concentrate DM, and factor C was level of the FCR at 0, 200, 300 and 400 mg DM of the total substrate. The study found that elemental sulfur, urea and FCR had no interaction effect on the kinetics of in vitro gas, ruminal fermentation, HCN and in vitro degradability. Elemental sulfur supplementation (P < 0.05) significantly increased the in vitro gas produced from an insoluble fraction (b), in vitro DM degradability and either neutral detergent fiber (NDF) or acid detergent fiber (ADF) degradability and propionate (C3) concentration while decreased the ruminal HCN concentration. Urea levels showed a (P < 0.05) significant increase of the potential extent of in vitro gas production, ruminal ammonia nitrogen (NH3-N) and total volatile fatty acid (TVFA). Fresh cassava root supplementation (P < 0.05) significantly increased the in vitro gas produced from an immediate soluble fraction (a), in vitro gas produced from insoluble fraction, in vitro gas production rate constant, total VFA, C3 concentration and HCN while decreased ruminal pH, acetate and butyrate concentration. It could be concluded that 2 % elemental sulfur, 4 % urea and 300 mg FCR showed a greater effect on in vitro gas production, ruminal fermentation and HCN reduction.ConclusionsThe study found that elemental sulfur, urea, and FCR had no interaction effect on the kinetics of in vitro gas, total in vitro gas, ruminal fermentation, and HCN concentration. It could be concluded that 2 % elemental sulfur, 4 % urea, and 300 mg FCR showed a greater effect on in vitro gas production, ruminal fermentation, and HCN reduction.

Hydrolysis of lignocellulosic forages by Trichoderma longibrachiatum isolate from bovine rumen

Fungi of the genus Trichoderma hydrolyze lignocellulosic materials, thus promoting the production of fermentable sugars. In the present study, the production of cellulases by Trichoderma longibrachiatum from ruminal fluid was evaluated in submerged fermentation of Urochloa decumbens hay (UD) or sugarcane bagasse (SB). Moreover, the biomass of this fungus was analyzed by supplementing it with urea or ammonium sulfate and with two buffer systems for the production of cellulases by T. longibrachiatum using SB or UD. The fermentations were carried out in test tubes containing Sabouraud broth, and the carbon source was packed in nonwoven fabric sachets. The fermentations were performed at 39 °C for 10 days. The factorial design was completely randomized, and the final pH, fungal biomass, substrate dry matter degradation, total protein, and cellulolytic enzymes were evaluated after the fermentation process. The fungal development was better in the medium supplemented with ammonium sulfate than that supplemented with urea, and it showed better degradation of UB than SB. The evaluated isolate promoted better protein synthesis during fermentation of SB buffered with sodium acetate; however, the medium containing UB produced higher concentrations of total cellulase, carboxymethylcellulase, and avicelase than the medium containing SB. Thus, rapid cellulose production by this fungus may aid in the hydrolysis of lignocellulosic materials for animal feed supplementation for ruminants; furthermore, the use of these enzymes for biotechnological purposes is promising.

In Vitro Fermentation Characteristics and Methane Mitigation Responded to Flavonoid Extract Levels from Alternanthera sissoo and Dietary Ratios

Two experiments were conducted under this study: Experiment 1 was to study production yield, chemical composition, and in vitro degradability of Brazilian spinach (Alternanthera sissoo; BS) leaf and leaf + leaf-stalk at various maturity ages of 15, 30, 45, and 60 days after plantation and regrowth and Experiment 2 was to evaluate the effect of flavonoid extract from BS leaf and leaf + leaf-stalk and dietary ratios on ruminal gas production, fermentation characteristics, and in vitro degradability. Experiment 1 showed that maturity ages after planting and regrowth increased, the yield significantly increased. Increasing maturity ages significantly (p &lt; 0.05) increased neutral detergent fiber and acid detergent fiber content and decreased crude protein content, total flavonoid (TF) content, and degradability for both leaf and leaf + leaf-stalk. Maturity ages from 15 to 30 days after plantation and regrowth resulted (p &lt; 0.05) the highest TF content and degradability for both leaf and leaf + leaf-stalk. Thus, BS leaf and leaf + leaf-stalk samples from 15 to 30 days of age were used for flavonoid extraction and used in the Experiment 2. Experiment 2 was conducted according to a 3 × 5 factorial experiment. Three roughage to concentrate (R:C) ratios at 50:50, 40:60, and 30:70 were used, and five levels of flavonoid extract (FE) at 0, 10, 20, 30, and 40 mg of substrate dry matter (DM) were supplemented. Experiment 2 showed that R:C ratio and FE had an interaction effect only on acetate to propionate ratio. Varying R:C ratios significantly increased (p &lt; 0.05) in vitro DM degradability, total volatile fatty acids (VFA), and propionate (C3) concentration. FE supplementation linearly (p &lt; 0.05) increased total VFA and C3 concentration and decreased methane production and protozoal population. This study could conclude that FE from BS could effectively modulate ruminal fermentation and decrease methane production. However, in vivo study needs to elucidate in order to validate the present results.

In-vivo anthelmintic activity assessment to dietary incorporation of natural feed additives in sheep: A comparative study

Anthelmintic activity to dietary incorporation of exogenous fibrolytic enzymes (EFE) and wormwood (Artemisia absinthium L.) herb as feed additives was evaluated. Twenty crossbred lambs (11.58±0.01 kg body weight) were distributed into four dietary treatments in completely randomized design and fed for a period of 90 days followed by 6 day digestibility trial. Animals in all the groups were offered oats straw based total mixed ration added with EFE’s cocktail at 0.60% substrate dry matter (DM) level (T1) or wormwood herb at 4.50% substrate DM level (T2) either alone, and in-combination of the two feed additives (T3), whereas the TMR without addition of any additive served as control (T0). In vivo anthelmintic assays and selected haemato-biochemical parameters of animals were evaluated at start (0d) and subsequently at monthly intervals (30, 60 and 90d). The feed additives in-combination improved nutrient digestibility and digestible nutrient intakes per day. Wormwood herb inclusion had significant positive effect on overall treatment means as well as at each feeding period of per cent faecal egg count reduction and at 90 day period of faecal parasitic eggs per gram assay. Blood profile revealed better physiological health status of lambs fed feed additives compared to those of control group. It is concluded that incorporation of wormwood had better anthelmintic activity and the two feed additives act synergistically in sheep to improve nutrient utilisation and gastrointestinal infested host’s health.

Maggot oil as a feed supplement for reducing methanogenesis of rumen microbial culture in vitro

Hermetia illucens larvae or known as maggot contains considerable amount of oil in its body. This experiment aimed to evaluate the use of maggot oil as a feed supplement for reducing methanogenesis of rumen microbial culture in vitro. The oil was supplemented into a feed substrate that consisted of forage: concentrate mixture (3:2 w/w). Supplementation of the oil was performed at different levels, i.e., 0, 1, 2, 3, 4 and 5% from substrate dry matter. All incubation bottles were tightly closed and incubated for 24 h in a water bath that maintained at 39oC. Results revealed that maggot oil at 4 and 5% supplementation level was able to reduce methane emission by 20.7 and 26.9% (P<0.05) in comparison to control, respectively. However, its supplementation at 3% or lower did not alter the methane emission. The organic matter digestibility parameter was linearly reduced with increasing level of maggot oil supplementation (P<0.05), but volatile fatty acid concentration was not affected. In contrast to our expectation, maggot oil at 3 to 5% increased log protozoa population and ammonia concentration than those of control (P<0.05). In conclusion, maggot oil may serve as a promising supplement for mitigating ruminal methanogenesis and the effect is dose-dependent.

PSXI-15 Effects of post-pyrolysis treated biochars on nutrient disappearance, methane production and ruminal fermentation of a silage-based diet in an artificial rumen system (RUSITEC)

Abstract This study investigated the effects of post-pyrolysis treated biochar on nutrient disappearance, total gas and methane (CH4) production, rumen fermentation and microbial protein synthesis in an artificial rumen system (RUSITEC) fed a barley silage-based diet. The basal diet consisted of 60% barley silage, 27% barley grain, 10% canola meal and 3% mineral/vitamin supplement (DM basis). Three spruced-based biochars, treated post-pyrolysis with either zinc chloride, hydrochloric acid/nitric acid mixture or sulfuric acid were added at 2.0% of substrate DM. In a randomized complete block design, treatments were assigned to sixteen vessels (n = 4/treatment) in two RUSITEC systems. The experiment was conducted over 15 d, with 8 d of adaptation and 7 d of sampling. Nutrient disappearance of dry matter (DM), organic matter (OM), acid detergent fiber (ADF) and neutral detergent fiber (NDF) was determined after 48 h of incubation from d 9 to 12, and microbial protein synthesis was measured from d 13–15. Data were analyzed using PROC MIXED in SAS, with the fixed effect of treatment and random effect of RUSITEC system and vessel. Biochar inclusion did not affect disappearance of DM (P = 0.49), OM (P = 0.60), CP (P = 0.14), NDF (P = 0.48), ADF (P = 0.11) or starch (P = 0.58). Biochar also had no effect on total gas production (P = 0.31) or CH4 produced expressed as a % of total gas production (P = 0.06), mg/d (P = 0.70), mg/g of DM incubated (P = 0.74), or mg/g of DM digested (P = 0.64). No effect on total VFA (P = 0.56) or NH3-N (P = 0.20) production were observed. Neither microbial protein synthesis nor total protozoa count were affected by biochar addition (P &amp;gt; 0.05). In conclusion, biochar inclusion in a silage-based diet did not exhibit the potential to mitigate CH4 emissions or improve digestion in a RUSITEC system.

In vitro fermentation of fodder beet root increases cumulative gas production of methane and carbon dioxide.

The relationship between proportion of fodder beet root (FB: Beta vulgaris subsp. vulgaris L.) in a perennial ryegrass (Lolium perenne) diet and gas production was investigated in vitro. Cumulative gas production and fermentation products were measured in calibrated glass syringes using a factorial arrangement (4 x 6), of dry matter proportion of FB (0, 15, 35 and 50% DM: 0FB, 15FB, 35FB and 50FB, respectively), and incubation time (0, 2, 4, 8, 12 and 24 h). Concentrations of methane (CH4) and carbon dioxide (CO2) produced both in fermentation and from bicarbonate buffering of volatile fatty acid, were also calculated. The proportion of FB root linearly increased volume of gas (mL) produced per g dry matter (DM) and per g organic matter (OM) with gas accumulation 14.8% greater for 50FB compared to 0FB at 24-hours of incubation (P<0.001). The concentration of propionic and butyric acids also increased relative to the proportion of FB (P < 0.001). Stoichiometric calculations indicate that FB increased CO2 released from both buffering and fermentation processes but reduced the fractions of methane in gas (17.6, 17.4, 17.0 and 16.8 ± 0.4% for 0FB, 15FB, 35FB, and 50FB, respectively, P< 0.001). However, the greater OM content of FB increased the total volume of both CO2 and CH4 produced. Results suggest that FB altered the thermodynamics of fermentation early in digestion by increasing formation of propionate which acted as a hydrogen sink to reduce formation of methane. However, as fermentation progressed, butyrate forming pathways became more favourable. The greater OM content of FB compared to ryegrass increased absolute volumes of CO2 and CH4 from fermentation, resulting in greater emission of gasses per kg DM of substrate. Further investigation in vivo is required to determine potential effects of microbial protein synthesis on gas production and further verify these results.

Demonstration‐scale enzymatic saccharification of sulfite‐pulped spruce with addition of hydrogen peroxide for LPMO activation

AbstractThe saccharification of lignocellulosic materials like Norway spruce is challenging due to the recalcitrant nature of the biomass, and it requires optimized and efficient pretreatment and enzymatic hydrolysis processes to make it industrially feasible. In this study, we report successful enzymatic saccharification of sulfite‐pulped spruce (Borregaard's BALI™ process) at demonstration scale, achieved through the controlled delivery of hydrogen peroxide (H2O2) for the activation of lytic polysaccharide monooxygenases (LPMOs) present in the cellulolytic enzyme preparation. We achieved 85% saccharification yield in 4 days using industrially relevant conditions – that is, an enzyme dose of 4% (w/w dry matter of substrate) of the commercial cellulase cocktail Cellic CTec3 and a substrate loading of 12% (w/w). Addition of H2O2 and the resulting controlled and high LPMO activity had a positive effect on the rate of saccharification and the final sugar titer. Clearly, the high LPMO activity was dependent on feeding the reactors with the LPMO co‐substrate H2O2, as in situ generation of H2O2 from molecular oxygen was limited. These demonstration‐scale experiments provide a solid basis for the use of H2O2 to improve enzymatic saccharification of lignocellulosic biomass at large industrial scale.© 2020 The Authors. Biofuels, Bioproducts, and Biorefining published by Society of Chemical Industry and John Wiley &amp; Sons, Ltd.

PSIX-12 Screening of peptides for their impact of protease, protease dose and peptide dose on in vitro rumen dry matter digestibility

Abstract The objective of this study was to screening peptides for the effects on in vitro rumen dry matter (DM) digestibility (DMD) of feeds in batch culture. Three brewers spent grain (BSG) samples from different batches were obtained from a local brewery. Peptides were developed with BSG protein varying protease (alcalase, everlase, flavourzyme) and protease dosages (protease:BSG protein; 1:100, 5:100, 10:100, 15:100) under optimum pH and temperature conditions. In total, 36 peptides were produced and screened. A series of batch cultures were conducted and substrates were barley silage and barley grain. The experiment was a complete randomized design with a factorial arrangement; 3 proteases × 4 protease doses + 4 doses of each peptide (0, 0.5, 1.0, 1.5% of substrate DM) with 3 replications of each treatment combination. Rumen inoculum was from three rumen fistulated beef heifers fed a diet consisting of 10% silage and 90% concentrate (DM basis). The incubation was conducted at 39°C for 24 h. Interactions among protease, protease dose and peptide dose were not significant. For barley grain, DMD differed (P &amp;lt; 0.01) among proteases (85.2, 72.3, 65.2%, respectively, for alcalase, flavourzise, everlase). Increasing peptide doses from 0, 0.5, 1.0 to 1.5% linearly (P &amp;lt; 0.01) decreased the DMD from 88 to 78% (alcalase), 69 to 59% (everlase) and 78 to 67% (flavourzyme); whereas protease dose did not affect the DMD. With barley silage, the DMD was higher (P &amp;lt; 0.05) with flavourzyme (65.3%) than alcalase (59.2%) or everlase (61.2%); in contrast, the DMD was affected neither by protease dose nor by the peptide dose, except that increasing supplementation of peptide derived from everlase linearly (P &amp;lt; 0.05) decreased the DMD (65.1 to 58.9%). These results indicated that the peptides derived from BSG decreased in vitro rumen feed digestibility, which varied with proteases, peptide dose or feeds.

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.

Comparison of lovastatin, citrinin and pigment production of different Monascus purpureus strains grown on rice and millet.

Six Monascus purpureus (red mould) strains were cultivated on brown rice and millet as substrates. They underwent strain selection for high lovastatin and pigment production, and low citrinin mycotoxin production, with particular reference to potential for millet as substrate. For most of these strains, substrate dry matter loss was 54-60% on rice and 46-48% on millet, although the 'MOPU GS1' strain showed 18% and 17% dry matter loss, respectively. 'MOPU GS1' was also the only strain with detectable levels of lovastatin (1.3 and 1.6mg lovastatin/g substrate dry weight [dw], respectively) and citrinin under the limit of detection. In the other strains, citrinin varied from 0.3 to 18.2mg/g substrate dw. Among the six strains, 'EBY3' provided high pigment production when grown on rice, although it produced 1.1mg citrinin/g substrate dw. Millet showed good potential as an alternative substrate to rice, due to higher lovastatin and lower citrinin production; however, rice was the better substrate for production of M. purpureus pigments.

Effect of Chinese herbal medicines on rumen fermentation, methanogenesis and microbial flora &lt;i&gt;in vitro&lt;/i&gt;

This study was carried out to evaluate the effects of three Chinese herbal medicines (CHMs) on rumen fermentation, methane emission and population of ruminal microbes using an in vitro gas production technique. Three healthy wethers of Dazu Black goats with similar bodyweights and permanent rumen fistulae were utilized as donors of ruminal fluid. The three botanical medicines were cablin patchouli herb (CPH), atractylodes rhizome (AR) and Amur cork tree (AC). Each CHM was added at a level of 25 g/kg to the substrate dry matter. In vitro gas production was recorded, and methane concentration was determined at 12 and 24 hours of incubation. After 24 hours, the incubation was stopped, and the inoculants were measured for pH, ammonia nitrogen and volatile fatty acids (VFAs) concentrations. Total deoxyribonucleic acid of ruminal microbes was extracted from the inocula, and populations were determined by a real-time quantitative polymerase chain reaction. Populations of total rumen methanogens, protozoa, total fungi, Ruminococcus albus , Fibrobacter succinogenes and Ruminococcus flavefaciens were expressed as a proportion of total rumen bacterial 16S ribosomal deoxyribonucleic acid. Compared to the control, CPH decreased gas production and methane production at 12 and 24 hours of incubation, and inhibited methanogens and total fungi growth. AR decreased acetate to propionate ratio, and methanogens and total fungi populations, but increased propionate molar proportion. AC decreased total VFA concentration, acetate to propionate ratio, gas production at 12 and 24 hours of incubation, methane production at 12 and 24 hours of incubation, and methanogens and total fungi growth, but increased the propionate molar proportion. In conclusion, CPH and AC both suppressed methanogenesis significantly, and the suppression was mediated primarily via the direct action against the rumen microbes involved in methane formation. AC also indirectly abates methane release by occupying the hydrogen (H 2 ) normally utilized for methanogenesis. Keywords: In vitro gas production, methane, rumen fermentation parameter, rumen microbes

Effect of Cobalt Chloride on Fermentation of Alfalfa and Smooth Bromegrass Hays by Horse Cecal Microorganisms

Effects of Co on fiber digestibility in horses are largely unknown. Our objective was to evaluate effects of Co chloride on in vitro gas production, VFA production, and dry matter (DM) disappearance (IVDMD) using cecal fluid from 4 cannulated Quarter Horses. Five grams DM of alfalfa or smooth bromegrass hay were provided as substrate with Co added at 0.0, 0.5, 5.0, 25.0, or 50.0 mg/kg substrate DM. Ten milliliters cecal fluid and 140 mL McDougall's buffer were incubated in duplicate, and pH, VFA concentrations, and IVDMD were measured after 48 hours. Gas production, used as an indicator of fermentative activity, was recorded every 15 minutes. Terminal pH was not affected by Co or forage type (P > .19). There was greater IVDMD in cultures containing alfalfa (29.2%) compared with smooth bromegrass hay (19.4%; P < .01). There was a forage × Co interaction (P < .05) in which gas production was greater in cultures containing alfalfa, and 5 mg Co/kg substrate DM led to greater gas production than 50 mg Co/kg substrate DM in cultures containing brome (P < .05). Gas production was affected by forage, Co, and time (P < .01). Production of individual and total VFA, as well as acetate:propionate (A:P) ratio were increased in cultures containing alfalfa (P < .05); however, there were no effects of Co on VFA concentrations (P > .05). While gas production was influenced by Co in cultures containing brome, no effects of Co were observed for pH, VFA, or IVDMD.