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Although 3-nitroxypropanol (3-NOP) has been proven to reduce enteric methane (CH 4 ) by ∼30% in indoor systems of dairying when the additive is mixed throughout a TMR, very limited research has been done to date in grazing systems in which the most convenient method of additive supplementation is at milking twice daily. To investigate the effect of twice daily 3-NOP supplementation on enteric CH 4 emissions, a 12-wk study was undertaken in which treatment cows (n = 26) were supplemented with 3-NOP (80 mg/kg DMI) twice daily at morning and evening milking, and control cows (n = 26) received no additive supplementation. Enteric CH 4 , hydrogen (H 2 ) and carbon dioxide (CO 2 ) were measured using GreenFeed units, and milk production, BW, BCS, and DMI were monitored to determine the effect of 3-NOP supplementation on productivity. No significant effect was observed for 3-NOP supplementation on any of the aforementioned parameters with the exception of CH 4 and H 2 production, respectively. Cows supplemented with 3-NOP produced 1.6-fold more H 2 (P < 0.001) across a 24-h period, with reductions in CH 4 production of 28.5% recorded in the 3 h after additive consumption (P < 0.001), however, levels of CH 4 production returned to that of the control group thereafter. When CH 4 production was considered across the entire 24-h period, the cows offered 3-NOP produced ∼5% less CH 4 than the control (P < 0.050). Future research should focus on methods to increase the efficacy of the additive throughout the day which would include the deployment of a slow-release form or an out-of-parlor feeding system that allows animals consume the product at additional time points., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective was to assess the supplementation with microencapsulation of hemp leaf extract (mHLE) utilized as a rumen enhancer on in vitro rumen fermentation and to enhance the bioavailability of active compounds for antimicrobial action, particularly in protozoa and methanogen populations. The feed treatments were totally randomized in the experimental design, with different levels of mHLE diet supplemented at 0, 4, 6 and 8% of total DM substrate and added to an R:C ratio of 60:40. During fermentation, gas kinetics production, nutrient degradability, ammonia nitrogen concentration, volatile fatty acid (VFA) profiles, methane production, and the microbial population were measured. The supplemented treatment at 6% of total DM substrate affected reductions in gas kinetics, cumulative gas production, and volatile fatty acid profiles, especially the acetate and acetate to propionate ratio. Whereas propionate proportion and total volatile fatty acid concentration were enhanced depending on the increase of nutrients in vitro dry matter degradability (IVDMD) after 12 h of post-fermentation at a R:C ratio of 60:40 (P < 0.05). Consequently, mHLE addition resulted in optimal ruminal pH and increased nutrient degradability, followed by ammonia nitrogen concentrations (P < 0.05), which were enhanced by dominant cellulolytic bacteria, particularly Ruminococcus albus and Ruminococcus flavefaciens, which showed the highest growth rates in the rumen ecology. Therefore, mHLE, a rich phytonutrient feed additive, affected the methanogen population, reduced the calculated methane production and can be a potential supplement in the ruminant diet., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Phupaboon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Methane is a short-lived greenhouse gas but has a far greater warming effect than carbon dioxide. At the same time, the livestock sector serves as a large contributor to global emissions of anthropogenic methane. Herein, this work aimed to use cultivated seaweed supplementation to reduce methane emissions and investigate the potential influencing mechanism. To evaluate the feasibility, two cultivated seaweeds, Laminaria japonica Aresch, and Porphyra tenera, along with the enzymatic hydrolysates derived from L. japonica, underwent in vitro trials, and they were both added into corn silage feed (CSF) with different concentrations (1%, 5%, and 10% of CSF) for methane reduction evaluation. The results indicated that >75% and 50% reductions in methane production were observed for the seaweeds and seaweed enzymatic hydrolysates in 9- and 30-day, respectively. Combined high-throughput sequencing and multivariate analysis revealed that supplementation with seaweed and seaweed enzymatic hydrolysates had a notable impact on the prokaryotic community structure. Mantel tests further revealed that significant correlations between the prokaryotic community and methane accumulation (P < 0.05), implying the prokaryotic community plays a role in reducing methane emissions within the rumen. Correspondingly, the networks within the prokaryotic community unveiled the crucial role of propionate/butyrate-producing bacteria in regulating methane emissions through microbial interactions. The predicted function of the prokaryotic community exhibited a significant reduction in the presence of the narB gene in seaweed-supplemented treatments. This reduction may facilitate an increased rate of electron flow toward the nitrate reduction pathway while decreasing the conversion of H 2 to methane. These results indicated the supplementation of cultivated seaweeds and the enzymatic hydrolysates has the potential to reshape the community structure of rumen microbial communities, and this alteration appears to be a key factor contributing to their methane production-reduction capability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This case study aimed to isolate and identify methanogenic bacteria from landfill soil, mud, and leachate samples to assess their role in anaerobic digestion and biogas production. Anaerobic digestion involves the breakdown of organic matter by a diverse group of bacteria under oxygen-free conditions, resulting in the production of methane and carbon dioxide. The collected samples from the landfill were cultured in a modified mineral salt medium (MSM). Microscopic observations revealed distinct coccus and bacillus morphologies of the isolated methanogenic bacteria. Gas production experiments and substrate utilization studies identified two types of methanogens. Methanosarcina sp., which utilized acetate and methanol for methane production, and Methanobacterium sp., utilizing hydrogen and carbon dioxide, as well as acetate. Scanning electron microscope (SEM) analysis confirmed the different morphotypes of the isolated methanogens. The study findings demonstrated the presence of diverse methanogens in the landfill environment, contributing to anaerobic digestion and biogas production.

Dietary carbohydrate manipulation can be used to reduce enteric CH 4 emission, but few studies have focused on the interaction of the different types of carbohydrates that can affect feed intake and ruminal fermentation. Understanding this interaction is necessary to make the most out of CH 4 mitigation feeding strategies using different dietary carbohydrates. The aim of this study was to test the effect on enteric CH 4 emission, feed intake, and milk production response when cows were fed either grass-clover silage (GCS) or corn silage (CS) as the sole forage source (55% of dry matter, DM), in combination with either barley (BAR) or dried beet pulp (DBP) as a concentrate (21.5% of DM). A total of 24 (half first-parity and half second-parity) cows were used in a crossover design with 2 periods of 21 d each, receiving 2 of 4 diets obtained from a 2 × 2 factorial arrangement of the experimental diet. Feed intake, CH 4 emission metrics, and milk production were recorded at the end of the experimental periods. The diets had NDF concentrations between 258 and 340 g/kg of DM and starch concentrations between 340 and 7.45 g/kg of DM (CS-BAR and GCS-DBP, respectively). The effects of silage and concentrate on dry matter intake (DMI) were additive, with the highest feed intake in cows fed CS-BAR, followed by cows fed CS-DBP, GCS-BAR, and GCS-DBP (21.2, 19.9, 19.1, and 18.3 kg/d, respectively). Energy corrected milk (ECM) yield was not affected by silage source in first parity cows, but it was higher for cows fed CS than cows fed GCS in second parity. The effects of silage and concentrate on CH 4 production (g/d), yield (g/kg of DMI), and intensity (g/kg of ECM) were not additive, as cows fed GCS had similar responses regardless of the concentrate used, but cows fed CS had lower CH 4 production, yield, and intensity when fed BAR instead of DBP. The lower CH 4 production, yield, and intensity in cows fed CS-BAR compared with other diets could be partially explained by the nonlinear relationship between ruminal VFA and carbohydrates (NDF and starch) concentration reported in the literature; however, we observed a linear relationship between the acetate/propionate ratio and CH 4 yield, suggesting possible other effects. The effects of silage and concentrate on the ruminal VFA were additive in first parity cows, but not in second parity cows. The interaction between dietary carbohydrate type and parity might indicate an effect of feed intake or the energy balance of the cow. Feeding cows silage and concentrate both rich in starch can result in the lowest enteric CH 4 emission., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objectives were to investigate the effect of feeding and visiting behavior of dairy cattle on CH 4 and H 2 production measured with voluntary visits to the GreenFeed system (GF) and to determine whether these effects depended on basal diet (BD) and 3-nitrooxypropanol (3-NOP) supplementation. The experiment involved 64 lactating dairy cattle (146 ± 45 DIM at the start of trial; mean ± SD) in 2 overlapping crossover trials, each consisting of 2 measurement periods. Cows within block were randomly allocated to 1 of 3 types of BD: a grass silage-based diet consisting of 30% concentrates and 70% grass silage (DM basis); a grass silage and corn silage mixed diet consisting of 30% concentrates, 42% grass silage, and 28% corn silage (DM basis); or a corn silage-based diet consisting of 30% concentrates, 14% grass silage, and 56% corn silage (DM basis). Each type of BD was subsequently supplemented with 0 and 60 mg 3-NOP/kg of DM in one crossover or 0 and 80 mg 3-NOP/kg of DM in the other crossover. Diets were provided in feed bins that automatically recorded feed intake and feeding behavior, with additional concentrate fed in the GF. All visits to the GF that resulted in a spot measurement of both CH 4 and H 2 emission were analyzed in relation to feeding behavior (e.g., meal size and time interval to preceding meal) as well as GF visiting behavior (e.g., duration of visit). Feeding and GF visiting behavior were related to CH 4 and H 2 production measured with the GF, in particular the meal size before a GF measurement and the time interval between a GF measurement and the preceding meal. Relationships between gas production and both feeding and GF visiting behavior were affected by type of BD as well as 3-NOP supplementation. With an increase of the time interval between a GF measurement and the preceding meal, CH 4 production decreased with 0 mg 3-NOP/kg of DM but increased with 60 and 80 mg 3-NOP/kg of DM, whereas type of BD did not affect these relationships. In contrast, CH 4 production increased with 0 mg 3-NOP/kg of DM but decreased with 60 and 80 mg 3-NOP/kg of DM upon an increase in the size of the meal preceding a GF measurement. With an increase of the time interval between a GF measurement and the preceding meal, or with a decrease of the size of the meal preceding a GF measurement, H 2 production decreased for all treatments, although the effect was generally somewhat stronger for 60 and 80 mg 3-NOP/kg of DM than for 0 mg 3-NOP/kg of DM. Hence, the timing of GF measurements next to feeding and GF visiting behavior are essential when assessing the effect of dietary treatment on the production of CH 4 and H 2 in a setting where a spot-sampling device such as a GF is used and where the measurements depend on voluntary visits from the cows., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Supplemental dietary rumen available fats show promise as enteric methane (eCH 4 ) mitigators for lactating dairy cows. However, concerns include variability in eCH 4 response and possible negative effects on dairy cow performance. Successful implementation of this mitigation option requires better prediction of responses specifically to rumen available fatty acids (FA) as well as understanding the modulating effects of other dietary and animal characteristics. Using meta-analytic and meta-regression techniques, 35 published studies with diet definition were used to assess changes in eCH 4 emissions and lactation performance associated with supplemental fat, specific supplemental rumen available FA types, and other dietary characteristics. Enteric CH 4 (g/d) was reduced by 3.77% per percentage unit of supplemental rumen available ether extract (EE). Supplemental rumen available PUFA (C18:2 and C18:3) and UFA (C18:1, C18:2, C18:3) mitigated eCH 4 (g/d) emissions in dairy cows by 6.88 and 4.65% per percentage unit increase, respectively. The anti-methanogenic effects of PUFA, MUFA and MCFA increased with correspondingly greater basal dietary levels of each FA type. Higher rumen-degradable starch (RDS; >18% DM) in the basal diet promoted greater reductions in eCH 4 yield (eCH 4 /DMI, g/kg) with supplemental rumen available PUFA and UFA. Both milk fat percentage and yield (kg/d) were reduced with rumen available fat supplementation with a reduction of 7.8% and 6.0%, respectively, relative to control diets. Our results highlight the importance of determining basal levels of the rumen available FA before providing supplemental rumen available FA as an option for enteric eCH 4 mitigation. Dairy nutritionists can use estimates generated from this analysis to predict changes in eCH 4 emissions and dairy cow performance associated with dietary supplementation of rumen available EE and specific rumen available FA types for the purpose of eCH 4 mitigation., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Dry anaerobic digestion (dry-AD) is an attractive process for solid wastes such as agri-food waste. However, some limitations mainly associated to lack of effective mixing, can hinder the methane production capacity of the systems. Bulking agent (BA) has been proposed as a solution to the compaction issues in systems without mechanical agitation, such as leaching bed reactors. However, effects of BA are still not clear, and, thus, the factors to consider for its dose has not been optimized yet. This work studies the effect of BA in dry-AD. Two substrates with different characteristics were proposed as models, bean peel as a lignocellulosic substrate and a mixture of food waste as a readily biodegradable substrate. Inert plastic rings were used as BA at different BA:S ratios. Assessed BA:S ratio did not affect the performance of methane production for the lignocellulosic waste, but it did significantly affect to the easily biodegradable substrate, showing up to a 28% of methane production increase. This result could be due to the presence of lignocellulosic compounds in the bean peel, behaving like a natural BA. In assays with an increased bed height, the compaction of the system was more severe, resulting in the rapid acidification of the processes. At these conditions, the positive effect of BA addition was more marked, allowing methane production and no acidification of the system. Thus, the addition of BA is a suitable strategy for improving methane production or stability in dry-AD systems without requiring the stirring of the systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study was performed to evaluate the effects of rye silage treated with sodium formate (Na-Fa) and lactic acid bacteria (LAB) inoculants on the ruminal fermentation characteristics, methane yield and energy balance in Hanwoo steers. Forage rye was harvested in May 2019 and ensiled without additives (control) or with either a LAB inoculant or Na-Fa. The LAB ( Lactobacillus plantarum ) were inoculated at 1.5 × 10 10 CFU/g fresh matter, and the inoculant was sprayed onto the forage rye during wrapping at a rate of 4 L/ton of fresh rye forage. Sixteen percent of the Na-Fa solution was sprayed at a rate of approximately 6.6 L/ton. Hanwoo steers (body weight 275 ± 8.4 kg ( n = 3, group 1); average body weight 360 ± 32.1 kg ( n = 3, group 2)) were allocated into two pens equipped with individual feeding gates and used in duplicated 3 × 3 Latin square design. The experimental diet was fed twice daily (09:00 and 18:00) during the experimental period. Each period comprised 10 days for adaptation to the pen and 9 days for measurements in a direct respiratory chamber. The body weights of the steers were measured at the beginning and at the end of the experiment. Feces and urine were collected for 5 days after 1 day of adaptation to the chamber, methane production was measured for 2 days, and ruminal fluid was collected on the final day. In the LAB group, the ratio of acetic acid in the rumen fluid was significantly lower ( p = 0.044) and the ratio of propionic acid in the rumen fluid was significantly higher ( p = 0.017). Methane production per DDMI of the Na-FA treatment group was lower than that of the other groups ( p = 0.052), and methane production per DNDFI of the LAB treatment group was higher than that of the other groups ( p = 0.056). The use of an acid-based additive in silage production has a positive effect on net energy and has the potential to reduce enteric methane emissions in ruminants., Competing Interests: Jayeon Kim is employed by Cargill Agri Purina Inc. and Geumhwi Bang is employed by Farmsco Co., Ltd. The authors declare that they have no competing interests except to Jayeon Kim and Geumhwi Bang., (© 2024 Choi et al.)

Rice cultivation boasts a rich historical legacy, serving as the primary sustenance for over 50% of the global population. However, the cultivation process gives rise to the emission of methane (CH 4 ) and nitrous oxide (N 2 O), two potent greenhouse gases. Notably, the global warming potential (GWP) of CH 4 and N 2 O surpasses CO 2 by 27-30 times and 273 times over 100 years, respectively. Addressing this environmental challenge necessitates exploring technical approaches and management strategies to curb gas emissions while sustaining rice yields. Several critical factors have been identified and analyzed for their potential to mitigate greenhouse gas production during rice cultivation. These include water management, fertilizer management, biochar application, cultivar selection, straw management, modified planting methods, and integration of new energy machinery. A comprehensive understanding and implementation of these methods can contribute significantly to achieving a dual objective: reducing emissions and maintaining optimal rice yields. Looking ahead, a synergistic integration of these diverse methods and management approaches holds promise for more effective results. Furthermore, the intricate water networks associated with rice cultivation should be carefully considered in the overall strategy. By adopting a holistic approach that addresses both emission reduction and sustainable water usage, the future of rice cultivation can be shaped to align with environmental stewardship and food security., Competing Interests: Declaration of Competing interest All the authors have read the manuscript and agreed to submit to this journal. We confirm the work has not been published before and is not being submitted to any other journal for publication. The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Automated measurements of the ratio of concentrations of methane and carbon dioxide, [CH 4 ]:[CO 2 ], in breath from individual animals (the so-called "sniffer technique") and estimated CO 2 production can be used to estimate CH 4 production, provided that CO 2 production can be reliably calculated. This would allow CH 4 production from individual cows to be estimated in large cohorts of cows, whereby ranking of cows according to their CH 4 production might become possible and their values could be used for breeding of low CH 4 -emitting animals. Estimates of CO 2 production are typically based on predictions of heat production, which can be calculated from body weight (BW), energy-corrected milk yield, and days of pregnancy. The objectives of the present study were to develop predictions of CO 2 production directly from milk production, dietary, and animal variables, and furthermore to develop different models to be used for different scenarios, depending on available data. An international dataset with 2,244 records from individual lactating cows including CO 2 production and associated traits, as dry matter intake (DMI), diet composition, BW, milk production and composition, days in milk, and days pregnant, was compiled to constitute the training dataset. Research location and experiment nested within research location were included as random intercepts. The method of CO 2 production measurement (respiration chamber [RC] or GreenFeed [GF]) was confounded with research location, and therefore excluded from the model. In total, 3 models were developed based on the current training dataset: model 1 ("best model"), where all significant traits were included; model 2 ("on-farm model"), where DMI was excluded; and model 3 ("reduced on-farm model"), where both DMI and BW were excluded. Evaluation on test dat sets with either RC data (n = 103), GF data without additives (n = 478), or GF data only including observations where nitrate, 3-nitrooxypropanol (3-NOP), or a combination of nitrate and 3-NOP were fed to the cows (GF+: n = 295), showed good precision of the 3 models, illustrated by low slope bias both in absolute values (-0.22 to 0.097) and in percentage (0.049 to 4.89) of mean square error (MSE). However, the mean bias (MB) indicated systematic overprediction and underprediction of CO 2 production when the models were evaluated on the GF and the RC test datasets, respectively. To address this bias, the 3 models were evaluated on a modified test dataset, where the CO 2 production (g/d) was adjusted by subtracting (where measurements were obtained by RC) or adding absolute MB (where measurements were obtained by GF) from evaluation of the specific model on RC, GF, and GF+ test datasets. With this modification, the absolute values of MB and MB as percentage of MSE became negligible. In conclusion, the 3 models were precise in predicting CO 2 production from lactating dairy cows., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Straw incorporation holds significant promise for enhancing soil fertility and mitigating air pollution stemming from straw burning. However, this practice concurrently elevates the production and emission of methane (CH 4 ) from paddy ecosystems. Despite its environmental impact, the precise mechanisms behind the heightened CH 4 production resulting from long-term straw incorporation remain elusive. In a 32-year field experiment featuring three fertilization treatments (CFS-chemical fertilizer with wheat straw, CF-chemical fertilizer, and CK-unamended), we investigated the impact of abiotic (soil physicochemical properties) and biotic (methanogenic abundance, diversity, and community composition) factors on CH 4 production in paddy fields. Results revealed a significantly higher CH 4 production potential under CFS treatment compared to CF and CK treatments. The partial least squares path model revealed that soil physicochemical properties (path coefficient = 0.61), methanogenic diversity (path coefficient = -0.43), and methanogenic abundance (path coefficient = 0.29) collectively determined CH 4 production potential, explaining 77% of the variance. Enhanced soil organic carbon content and water content, resulting from straw incorporation, emerged as pivotal factors positively correlated with CH 4 production potential. Under CFS treatment, lower Shannon index of methanogens, compared to CF and CK treatments, was attributed to increased Methanosarcina. Notably, the Shannon index and relative abundance of Methanosarcina exhibited negative and positive correlations with CH 4 production potential, respectively. Methanogenic abundance, bolstered by straw incorporation, significantly amplified overall potential. This comprehensive analysis underscores the joint influence of abiotic and biotic factors in regulating CH 4 production potential during multi-decadal straw incorporation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Conductive materials (CM) enhance methanogenesis, but there is no clear correlation between conductivity and faster methane production (MP) rates. We investigated if MP by pure cultures of methanogens (Methanobacterium formicicum, Methanospirillum hungatei, Methanothrix harundinacea and Methanosarcina barkeri) is affected by CM (activated carbon (AC), magnetite), and other sustainable alternatives (sand and glass beads, without conductivity, and zeolites (Zeo)). The significant impact of the materials was on M. formicicum as MP was significantly accelerated by non-CM (e.g., sand reduced the lag phase (LP) duration by 48 %), Zeo and AC (LP reduction in 71% and 75 %, respectively). Conductivity was not correlated with LP reduction. Instead, silicon content in the materials was inversely correlated with the time required for complete MP, and silicon per se stimulated M. formicicum's activity. These findings highlight the potential of using non-CM silicon-containing materials in anaerobic digesters to accelerate methanogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The objective of this study was to determine the potential effect and interaction of 3-nitrooxypropanol (3-NOP; Bovaer, DSM-Firmenich Nutrition Products Ltd.) and whole cottonseed (WCS) on lactational performance and enteric methane (CH 4 ) emission of dairy cows. A total of 16 multiparous cows, including 8 Holstein Friesian (HF) and 8 Brown Swiss (BS; 224 ± 36 DIM, 26 ± 3.7 kg milk yield, mean ± SD), were used in a split-plot design, where the main plot was the breed of cows. Within each subplot, cows were randomly assigned to a treatment sequence in a replicated 4 × 4 Latin square design with 2 × 2 factorial arrangements of treatments with four 24-d periods. The experimental treatments were as follows: (1) control (basal TMR), (2) 3-NOP (60 mg/kg TMR DM), (3) WCS (5% TMR DM), and (4) 3-NOP + WCS. The treatment diets were balanced for ether extract, crude protein, and NDF contents (4%, 16%, and 43% of TMR DM, respectively). The basal diets were fed twice daily at 0800 and 1800 h. Dry matter intake and milk yield were measured daily, and enteric gas emissions were measured (using the GreenFeed System, C-Lock Inc.) during the last 3 d of each 24-d experimental period when animals were housed in tiestalls. There was no difference in DMI on treatment level, whereas the WCS treatment increased ECM yield and milk fat yield. No interaction of 3-NOP and WCS occurred for any of the enteric gas emission parameters, but 3-NOP decreased CH 4 production (g/d), CH 4 yield (g/kg DMI), and CH 4 intensity (g/kg ECM) by 13%, 14%, and 13%, respectively. Further, an unexpected interaction of breed by 3-NOP was observed for different enteric CH 4 emission metrics: HF cows had a greater CH 4 mitigation effect compared with BS cows for CH 4 production (g/d; 18% vs. 8%), CH 4 intensity (g/kg milk yield; 19% vs. 3%), and CH 4 intensity (g/kg ECM; 19% vs. 4%). Hydrogen production was increased by 2.85-fold in HF and 1.53-fold in BS cows receiving 3-NOP. Further, a 3-NOP × time interaction occurred for both breeds. In BS cows, 3-NOP tended to reduce CH 4 production by 18% at approximately 4 h after morning feeding, but no effect was observed at other time points. In HF cows, the greatest mitigation effect of 3-NOP (29.6%) was observed immediately after morning feeding, and it persisted at around 23% to 26% for 10 h until the second feed provision, and 3 h thereafter, in the evening. In conclusion, supplementing 3-NOP at 60 mg/kg DM to a high-fiber diet resulted in 18% to 19% reduction in enteric CH 4 emission in Swiss HF cows. The lower response to 3-NOP by BS cows was unexpected and has not been observed in other studies. These results should be interpreted with caution due to the low number of cows per breed. Finally, supplementing WCS at 5% of DM improved ECM and milk fat yield but did not enhance the CH 4 inhibition effect of 3-NOP of dairy cows., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Considering that the Brazilian southeast has several agricultural farms that produce coffee and raise swine, and that the waste generated in these farms has some complementary characteristics, the present study aimed to optimize the methane (CH 4 ) yield in the batch anaerobic co-digestion of liquid swine manure (LSM), coffee wastewater (CFW), and coffee husk and pulp. The optimization occurred through a two-factor central composite rotational design with a variation of CFW percentage (8 to 22 %) in the mixing liquid substrates and the organic matter concentration (0.3 to 12 gCOD L -1 ). The optimized condition had an ideal nutritional condition (14 % of CFW, 86 % of LSM, 7.3 gCOD L -1 and COD/N ratio of 35) to obtain high CH 4 production (971.7 mLCH 4 ), yield (160.9 mLCH 4 g -1 VS), maximum specific production rate (1.6 mL h -1 ) and low lag phase (217.6 h)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Accurate modeling of methane (CH 4 ) and sulfide (H 2 S) production in sewer systems was constrained by insufficient consideration of microbial processes under dynamic environmental conditions. This study introduces a microbial-guided machine learning (ML) framework (Micro-ML), which integrates microbial process representations from mechanistic models (microbial information) with ML models. Results indicate that Micro-ML model enhanced predictions of CH 4 and H 2 S production, where microbial information provides more information for model optimization. The feature importance of microbial information performed comparable weightings for 58.12 % and 55.16 %, respectively, but their relative significance in influencing Micro-ML model performance varies considerably. The application of Micro-ML performed great potential in reducing CH 4 and H 2 S production (decreased ∼ 80 % and 90 %). The integrated model not only improves the accuracy of CH 4 and H 2 S predictions but also offers a valuable tool for effective management strategies for sewer systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Coccomyxa sp. KJ is a unicellular green microalga that accumulates abundant lipids when cultured under nitrogen-deficient conditions (KJ1) and high nitrogen levels when cultured under nitrogen-sufficient conditions (KJ2). Considering the different characteristics between KJ1 and KJ2, they are expected to have different effects on rumen fermentation. This study aimed to determine the effects of KJ1 and KJ2 on in vitro ruminal fermentation, digestibility, CH4 production, and the ruminal microbiome as corn silage substrate condition. Five treatments were evaluated: substrate only (CON) and CON + 0.5% dry matter (DM) KJ1 (KJ1&#95;L), 1.0% DM KJ1 (KJ1&#95;H), 0.5% DM KJ2 (KJ2&#95;L), and 1.0% DM KJ2 (KJ2&#95;H). DM degradability-adjusted CH4 production was inhibited by 48.4 and 40.8% in KJ2&#95;L and KJ2&#95;H, respectively, compared with CON. The proportion of propionate was higher in the KJ1 treatments than the CON treatment and showed further increases in the KJ2 treatments. The abundances of Megasphaera, Succiniclasticum, Selenomonas, and Ruminobacter, which are related to propionate production, were higher in KJ2&#95;H than in CON. The results suggested that the rumen microbiome was modified by the addition of 0.5-1.0% DM KJ1 and KJ2, resulting in increased propionate and reduced CH4 production. In particular, the KJ2 treatments inhibited ruminal CH4 production more than the KJ1 treatments. These findings provide important information for inhibiting ruminal CH4 emissions, which is essential for increasing animal productivity and sustaining livestock production under future population growth., Competing Interests: Hitoshi Kuno worked for a company that produced the Coccomyxa sp. KJ, and partly funded the project. The other authors declare no conflicts of interest., (Copyright: © 2024 Sato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Methane is the second most abundant climate-active gas, and understanding its sources and sinks is an important endeavour in microbiology, biogeochemistry, and climate sciences 1,2 . For decades, it was thought that methanogenesis, the ability to conserve energy coupled to methane production, was taxonomically restricted to a metabolically specialized group of archaea, the Euryarchaeota 1 . The discovery of marker genes for anaerobic alkane cycling in metagenome-assembled genomes obtained from diverse habitats has led to the hypothesis that archaeal lineages outside the Euryarchaeota are also involved in methanogenesis 3-6 . Here we cultured Candidatus Methanosuratincola verstraetei strain LCB70, a member of the archaeal class Methanomethylicia (formerly Verstraetearchaeota) within the phylum Thermoproteota, from a terrestrial hot spring. Growth experiments combined with activity assays, stable isotope tracing, and genomic and transcriptomic analyses demonstrated that this thermophilic archaeon grows by means of methyl-reducing hydrogenotrophic methanogenesis. Cryo-electron tomography revealed that Ca. M. verstraetei are coccoid cells with archaella and chemoreceptor arrays, and that they can form intercellular bridges connecting two to three cells with continuous cytoplasm and S-layer. The wide environmental distribution of Ca. M. verstraetei suggests that they might play important and hitherto overlooked roles in carbon cycling within diverse anoxic habitats., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Methanogenesis mediated by archaea is the main source of methane, a strong greenhouse gas, and thus is critical for understanding Earth's climate dynamics. Recently, genes encoding diverse methanogenesis pathways have been discovered in metagenome-assembled genomes affiliated with several archaeal phyla 1-7 . However, all experimental studies on methanogens are at present restricted to cultured representatives of the Euryarchaeota. Here we show methanogenic growth by a member of the lineage Korarchaeia within the phylum Thermoproteota (TACK superphylum) 5-7 . Following enrichment cultivation of 'Candidatus Methanodesulfokora washburnenis' strain LCB3, we used measurements of metabolic activity and isotope tracer conversion to demonstrate methanol reduction to methane using hydrogen as an electron donor. Analysis of the archaeon's circular genome and transcriptome revealed unique modifications in the energy conservation pathways linked to methanogenesis, including enzyme complexes involved in hydrogen and sulfur metabolism. The cultivation and characterization of this new group of archaea is critical for a deeper evaluation of the diversity, physiology and biochemistry of methanogens., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Methanogenic archaea are main contributors to methane emissions, and have a crucial role in carbon cycling and global warming. Until recently, methanogens were confined to Euryarchaeota, but metagenomic studies revealed the presence of genes encoding the methyl coenzyme M reductase complex in other archaeal clades 1-4 , thereby opening up the premise that methanogenesis is taxonomically more widespread. Nevertheless, laboratory cultivation of these non-euryarchaeal methanogens was lacking to corroborate their potential methanogenic ability and physiology. Here we report the isolation of a thermophilic archaeon LWZ-6 from an oil field. This archaeon belongs to the class Methanosuratincolia (originally affiliated with 'Candidatus Verstraetearchaeota') in the phylum Thermoproteota. Methanosuratincola petrocarbonis LWZ-6 is a strict hydrogen-dependent methylotrophic methanogen. Although previous metagenomic studies speculated on the fermentative potential of Methanosuratincolia members, strain LWZ-6 does not ferment sugars, peptides or amino acids. Its energy metabolism is linked only to methanogenesis, with methanol and monomethylamine as electron acceptors and hydrogen as an electron donor. Comparative (meta)genome analysis confirmed that hydrogen-dependent methylotrophic methanogenesis is a widespread trait among Methanosuratincolia. Our findings confirm that the diversity of methanogens expands beyond the classical Euryarchaeota and imply the importance of hydrogen-dependent methylotrophic methanogenesis in global methane emissions and carbon cycle., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Controlled studies have extensively documented that concentrate supplements typically increase enteric CH 4 emissions and milk yield and reduce emissions per unit of milk produced and dry matter intake. However, no studies have been conducted to determine the effect of concentrate on predicted greenhouse gas emissions from dairy farms representing the Australian pasture-based farming system. Thus, this study sought to determine how dietary concentrate supplementation affects enteric and manure CH 4 , and N 2 O of Australian pasture-based dairy farms. The Australian Dairy Carbon Calculator was used, which incorporates emission factors and methodologies used in the National Greenhouse Gas Inventory as reported to the Intergovernmental Panel on Climate Change. Primary data were collected and analyzed from 120 commercial farms in Australia's major dairy regions. Then the farms were divided into 4 groups based on their dietary concentrate supplementation: ≤1 (low; 15 farms), 1 to 2 (moderate; 35 farms), 2 to 3 (high; 35 farms), and ≥3 (very high; 35 farms) t of concentrate dry matter (tDM) per cow per year. Sources of greenhouse gas emissions were CO 2 from concentrate production, enteric CH 4 , and manure CH 4 and N 2 O. Total dry matter intake, milk yield, and daily enteric CH 4 production (g/d) quadratically increased with concentrate level, whereas greenhouse gas emission intensity of milk production (kg of CO 2 equivalent per kg of fat- and protein-corrected milk) decreased by 14% for farms supplementing with ≥3 tDM/cow per year compared with those supplementing with ≤1 tDM/cow per year of dietary concentrate. The N 2 O and CH 4 emissions from manure increased quadratically and linearly, respectively, with the increasing supplementation of concentrate. Farms supplementing 2 to 3 tDM/cow per year showed substantial increases in gross income, gross margin, earnings before interest and tax, and net income ($/cow per year) compared with those supplementing of ≤1, 1 to 2, and ≥3 tDM/cow per year. Overall, increasing dietary concentrate supplementation for dairy cows resulted in increased milk production per cow, reduced greenhouse gas emissions per unit of milk produced, and increased income and profit. However, a comprehensive life cycle assessment study is needed to account for carbon sequestration by other farm components, such as pastures and trees, which were not considered in the present study. In addition, the present study was based on modeling and did not gather ground truth information for DMI, digestibility, crude protein, and urinary and fecal N excretion. Therefore, data should be interpreted with caution, and studies gathering such information are encouraged., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective was to determine the long-term effect of 3-nitrooxypropanol (3-NOP) on CH 4 emission and milk production characteristics from dairy cows receiving 3-NOP in their diet for a full year, covering all lactation stages of the dairy cows. Sixty-four late-lactation Holstein-Friesian cows (34% primiparous) were blocked in pairs, based on expected calving date, parity, and daily milk yield. The experiment started with an adaptation period of 1 wk followed by a covariate period of 3 wk in which all cows received the same basal diet and baseline measurements were performed. Directly after, cows within a block were randomly allocated to 1 of 2 dietary treatments: a diet containing on average 69.8 mg 3-NOP/kg DM (total ration level, corrected for intake of nonsupplemented GreenFeed bait) and a diet containing a placebo. Forage composition as well as forage-to-concentrate ratio altered with lactation stage (i.e., dry period and early, mid, and late lactation). Diets were provided as a total mixed ration, and additional bait was fed in GreenFeed units (C-Lock Inc.), which were used for emission measurements. Supplementation of 3-NOP did not affect total DMI, BW, or BCS, but resulted in a 6.5% increase in the yields of energy-corrected milk and fat- and protein-corrected milk (FPCM). Furthermore, milk fat and protein as well as feed efficiency were increased upon 3-NOP supplementation. Overall, a reduction of 21%, 20%, and 27% was achieved for CH 4 production (g/d), yield (g/kg DMI), and intensity (g/kg FPCM), respectively, upon 3-NOP supplementation. The CH 4 mitigation potential of 3-NOP was affected by the lactation stage dependent diet to which 3-NOP was supplemented. On average, a 16%, 20%, 16%, and 26% reduction in CH 4 yield (g/kg DMI) was achieved upon 3-NOP supplementation for the dry period, and early, mid, and late-lactation diets, respectively. The CH 4 mitigation potential of 3-NOP was affected by the length of 3-NOP supplementation within a lactation stage dependent diet and by variation in diet composition within a lactation stage dependent diet as a result of changes in grass and corn silage silos. In conclusion, 3-NOP reduced CH 4 emission from cows receiving 3-NOP for a year, with a positive effect on production characteristics. The CH 4 mitigation potential of 3-NOP was influenced by diet type, diet composition, and nutrition value, and the efficacy of 3-NOP appeared to decline over time but not continuously. Associated with changes in diet composition, increased efficacy of 3-NOP was observed at the start of the trial, at the start of a new lactation, and, importantly, at the end of the trial. These results suggest that diet composition has a large effect on the efficacy of 3-NOP, perhaps even larger than the week of supplementation after first introduction of 3-NOP. More studies are needed to clarify the long-term effects of 3-NOP on CH 4 emission and to further investigate what influence variation in diet composition may have on the mitigation potential of 3-NOP., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Greenhouse gas emission from the activities of all productive sectors is currently a topic of foremost importance. The major contributors in the livestock sector are ruminants, especially dairy cows. This study aimed to evaluate and compare 21 equations for predicting enteric methane emissions (EME) developed on the basis of milk traits and fatty acid profiles, which were selected from 46 retrieved through a literature review. We compiled a reference database of the detailed fatty acid profiles, determined by GC, of 992 lactating cows from 85 herds under 4 different dairy management systems. The cows were classified according to DIM, parity order, and dairy system. This database was the basis on which we estimated EME using the selected equations. The EME traits estimated were methane yield (20.63 ± 2.26 g/kg DMI, 7 equations), methane intensity (16.05 ± 2.76 g/kg of corrected milk, 4 equations), and daily methane production (385.4 ± 68.2 g/d, 10 equations). Methane production was also indirectly calculated by multiplying the daily corrected milk yield by the methane intensity (416.6 ± 134.7 g/d, 4 equations). We also tested for the effects of DIM, parity, and dairy system (as a correction factor) on the estimates. In general, we observed little consistency among the EME estimates obtained from the different equations, with exception of those obtained from meta-analyses of a range of data from different research centers. We found all the EME predictions to be highly affected by the sources of variation included in the statistical model: DIM significantly affected the results of 19 of the 21 equations, and parity order influenced the results of 13. Different patterns were observed for different equations with only some of them in accordance with expectations based on the cow's physiology. Finally, the best predictions of daily methane production were obtained when a measure of milk yield was included in the equation or when the estimate was indirectly calculated from daily milk yield and methane intensity., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to evaluate the interaction of dietary carbohydrate profile and soybean meal (SBM) replacement with either Chlorella pyrenoidosa (CHL) or Spirulina platensis (SPI) on in vitro fermentation. This experiment was conducted as a randomized complete block design, with fermentation run (3 runs) considered as blocks. The treatments were arranged in a 2 × 5 factorial design, where the first factor was the carbohydrate profile, which was composed of diets containing 42.5% neutral detergent fiber (NDF) and 26.8% starch (HF-LS) or 26.8% NDF and 40.6% starch (LF-HS), and the second factor was the protein source, in which a control diet (100% SBM), partial replacement of SBM with CHL (1/2CHL) or SPI (1/2SPI), or total replacement of SBM with CHL or SPI were used. All experimental diets were formulated to have 17% crude protein. The ruminal fluid was collected from 2 lactating Holstein cows, buffered with Van Soest medium at a ratio of 1:2 and added to serum bottles containing 0.50 g of the experimental diets. Bottles were incubated at 39°C for 24 and 48 h in triplicate; headspace pressure was measured, along with gas collection for methane (CH 4 ) quantification at 0, 2, 4, 8, 16, 24, 36, and 48 h after incubation. The final medium was used to measure pH, ammonia, and VFA. After incubation, feed bags were recovered and used for estimation of dry matter (DM), NDF, and organic matter (OM) degradability. Statistical analysis was carried out using the MIXED procedure of SAS, with carbohydrate profile, protein source, assay, and their interactions as fixed effects, with run and bottle as random effects. Orthogonal contrasts were used to compare carbohydrate profile, algae species, carbohydrate profile × algae interaction, and linear and quadratic effects of SBM replacement with CHL or SPI. There was no interaction effect between carbohydrate profile and algae source. The LF-HS improved gas production, degradability of nutrients, and VFA, mainly increasing the production of butyrate and propionate. When compared with CHL, SPI had a greater degradability of nutrients and branched VFA, along with reduction in total gas production and tended to reduce total CH 4 yield. The replacement of SBM with algae linearly reduced the degradability of nutrients, along with a linear reduction in gas production. When replacement of SBM with only SPI was evaluated, SPI slightly reduced the degradability of nutrients; however, it promoted a linear reduction in CH 4 yield, as well as reduction in CH 4 yield by unit of degraded DM, NDF, and OM. In summary, there was no interaction of carbohydrate profile and protein source, which means that SBM replacement had a similar effect, regardless of dietary carbohydrate profile. Spirulina may be a more suitable algae source than Chlorella due to the potential to reduce CH 4 ., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The large-scale recording of traits such as feed efficiency (FE) and methane emissions (ME) for use in genetic improvement programs is complex, costly, and time-consuming. Therefore, heritable traits that can be continuously recorded in dairy herds and are correlated with FE and ME traits could provide useful information for genetic evaluation. Rumination time has been suggested to be associated with FE, methane production (MeP; ME in g/d), and production traits at the phenotypic level. Therefore, the objective of this study was to investigate the genetic relationships among rumination time (RT), FE, methane and production traits using 7,358 records from 656 first-lactation Holstein cows. The estimated heritabilities were moderate for RT (0.45 ± 0.14), MeP (0.36 ± 0.12), milk yield (0.40 ± 0.08), fat yield (0.29 ± 0.06), protein yield (0.32 ± 0.07), and energy-corrected milk (0.28 ± 0.07), but were low and nonsignificant for FE (0.15 ± 0.07), which was defined as the residual of the multiple linear regression of DMI on energy-corrected milk and metabolic body weight. A favorable negative genetic correlation was estimated between RT and MeP (-0.53 ± 0.24), whereas a positive favorable correlation was estimated between RT and energy-corrected milk (0.49 ± 0.11). The estimated genetic correlation of RT with FE (-0.01 ± 0.17) was not significantly different from zero but showed a trend of a low correlation with dry matter intake (0.21 ± 0.13). These results indicate that RT is genetically associated with MeP and milk production traits, but high standard errors indicate that further analyses should be conducted to verify these findings when more data for RT, MeP, and FE become available., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Graphene oxide (GO) addition to anaerobic digestion has been suggested to enhance direct electron transfer. The impact of GO (0.075 g GO g -1 VS) and biologically and hydrothermally reduced GO (bio-rGO and h-rGO, respectively) on the methane production kinetics and removal of 12 pharmaceuticals was assessed in Fed-batch reactors. A decrease of 15 % in methane production was observed in the tests with GO addition compared with the control and the h-rGO. However, bio-rGO and h-rGO substantially increased the methane production rate compared to the control tests (+40 %), in the third fed-batch test. Removal of pharmaceuticals was enhanced only during the bio-reduction of GO (1st fed-batch test), whereas once the GO was bio-reduced, it followed a similar trend in the control and h-rGO tests. The addition of GO can enhance the methane production rate and, therefore, reduce the anaerobic treatment time., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Methane, both enteric and from manure management, is the most important greenhouse gas from ruminant livestock, and its mitigation can deliver substantial decreases in the carbon footprint of animal products and potentially contribute to climate change mitigation. Although choices may be limited, certain feeding-related practices can substantially decrease livestock enteric CH 4 emission. These practices can be generally classified into 2 categories: diet manipulation and feed additives. Within the first category, selection of forages and increasing forage digestibility are likely to decrease enteric CH 4 emission, but the size of the effect, relative to current forage practices in the United States dairy industry, is likely to be minimal to moderate. An opportunity also exists to decrease enteric CH 4 emissions by increasing dietary starch concentration, but interventions have to be weighed against potential decreases in milk fat yield and farm profitability. A similar conclusion can be made about dietary lipids and oilseeds, which are proven to decrease CH 4 emission but can also have a negative effect on rumen fermentation, feed intake, and milk production and composition. Sufficient and robust scientific evidence indicates that some feed additives, specifically the CH 4 inhibitor 3-nitrooxypropanol, can substantially reduce CH 4 emissions from dairy and beef cattle. However, the long-term effects and external factors affecting the efficacy of the inhibitor need to be further studied. The practicality of mass-application of other mitigation practices with proven short-term efficacy (i.e., macroalgae) is currently unknown. One area that needs more research is how nutritional mitigation practices (both diet manipulation and feed additives) interact with each other and whether there is synergism among feed additives with different mode of action. Further, effects of diet on manure composition and greenhouse gas emissions during storage (e.g., emission trade-offs) have not been adequately studied. Overall, if currently available mitigation practices prove to deliver consistent results and novel, potent, and safe strategies are discovered and are practical, nutrition alone can deliver up to 60% reduction in enteric CH 4 emissions from dairy farms in the United States., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Enteric CH 4 produced from dairy cows contributes to the emission of greenhouse gases from anthropogenic sources. Recent studies have shown that the selection of lower CH 4 -emitting cows is possible, but doing so would be simpler if performance measures already recorded on farm could be used, instead of measuring gas emissions from individual cows. These performance measures could be used for selection of low emitting cows. The aim of this analysis was to quantify how much of the between-cow variation in CH 4 production can be explained by variation in performance measures. A dataset with 3 experiments and a total of 149 lactating dairy cows with repeated measures was used to estimate the between-cow variation (the variation between cow estimates) for performance and gas measures from GreenFeed (C-Lock, Rapid City, SD). The cow estimates were obtained with a linear mixed model with the diet within period effect as a fixed effect and the cow within experiment as a random effect. The cow estimates for CH 4 production were first regressed on the performance and gas measures individually, and then performance and CO 2 production measures were grouped in 3 subsets for principal component analysis and principal component regression. The variables that explained most of the between-cow variation in CH 4 production were DMI (R 2 = 0.44), among the performance measures, and CO 2 production (R 2 = 0.61), among gas measures. Grouping the measures increased the R 2 to 0.53 when only performance measures were used, and to 0.66 when CO 2 production was added to the significant performance measures. We found the marginal improvement to be insufficient to justify the use of grouped measures rather than an individual measure because the latter simplifies the model and avoids over-fitting. Investigation of other measures that can be explored to increase explanatory power of between-cow variation in CH 4 production is briefly discussed. Finally, the use of residual CH 4 as a measure for CH 4 efficiency could be considered by using either DMI or CO 2 production as the sole predicting variables., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

A large amount of agricultural waste causes global environmental pollution. Biogas production by microbial pretreatment is an important way to utilize agricultural waste resources. In this study, Sporocytophaga CG-1 (A, cellulolytic strain) was co-cultured with Bacillus clausii HP-1 (B, non-cellulolytic strain) to analyze the effect of pretreatment of rice straw on methanogenic capacity of anaerobic digestion (AD). The results showed that weight loss rate of filter paper of co-culture combination is 53.38%, which is 29.37% higher than that of A. The synergistic effect of B on A can promote its degradation of cellulose. The cumulative methane production rate of the co-culture combination was the highest (93.04 mL/g VS substrate), which was significantly higher than that of A, B and the control group (82.38, 67.28 and 67.70 mL/g VS substrate). Auxiliary bacteria can improve cellulose degradation rate by promoting secondary product metabolism. These results provide data support for the application of co-culture strategies in the field of anaerobic digestion practices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study proposes the was to evaluate the stability and methane production with organic load differents in an upflow anaerobic sludge blanket reactor (UASB) treating swine wastewater by methods of multivariate analysis. Four organic loads were used with average hydraulic holding times of one day. The methods of data analysis of linear regression, Pearson correlation, principal component analysis and hierarchical clustering analysis were used for understanding stability and methane production in the reactor. The highest concentrations of bicarbonate alkalinity of 683 mg L -1 CaCO 3 and total volatile acids of 1418 mg L -1 HAc with maximum organic loading applied were obtained. The optimal stability conditions occurred at an intermediate and partial alkalinity ratio between 0.24 and 0.25 observed in initial phases with a chemical oxygen demand (COD) removal of 47-57%. Maximum methane production was 9.0 L CH 4 d -1 observed with linear regression positive and occurred at the highest applied organic load, corresponding to the highest COD removal efficiency and increased microbial biomass. Positive and negative correlation between functional stability in anaerobic digestion showed regular activity between acids, alkalinity and organic matter removal. This fact was also proven by the analysis of principal components that showed three components responsible for explaining 83.2% of the data variability, and the alkalinity, organic matter influent and organic acids had the greatest effects on the stability of the UASB reactor. Hierarchical clusters detected the formation of five groupings with a similarity of 50.1%, indicating that temperature and pH were variables with unitary influences on data dimensionality., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

This study aimed to evaluate the effects of essential oils (EOs) extracted from Cannabis sativa L. and Cannabis indica Lam. on in vitro ruminal fermentation characteristics, selected rumen microbial populations, and methane production. GC-MS analyses allowed us to identify 89 compounds in both EOs. It was found that E -β-caryophyllene predominated in C. sativa (18.4%) and C. indica (24.1%). An in vitro (Ankom) test was performed to analyse the control and monensin groups, as well as the 50 µL or 100 µL EOs. The samples for volatile fatty acids (VFAs), lactate, and microbiological analysis were taken before incubation and after 6 and 24 h. The application of EOs of C. indica resulted in an increase in the total VFAs of acetate and propionate after 6 h of incubation. The applied EOs had a greater impact on the reduction in methane production after 6 h, but no apparent effect was noted after 24 h. Lower concentrations of C. sativa and C. indica had a more pronounced effect on Lactobacillus spp. and Buryrivibrio spp. than monensin. The presented findings suggest that C. sativa and C. indica supplementation can modify ruminal fermentation, the concentrations of specific volatile fatty acids, and methane production.

Ruminant livestock produce around 24% of global anthropogenic methane emissions. Methanogenesis in the animal rumen is significantly inhibited by bromoform, which is abundant in seaweeds of the genus Asparagopsis . This has prompted the development of livestock feed additives based on Asparagopsis to mitigate methane emissions, although this approach alone is unlikely to satisfy global demand. Here we engineer a non-native biosynthesis pathway to produce bromoform in vivo with yeast as an alternative biological source that may enable sustainable, scalable production of bromoform by fermentation. β-dicarbonyl compounds with low p K a values were identified as essential substrates for bromoform production and enabled bromoform synthesis in engineered Saccharomyces cerevisiae expressing a vanadate-dependent haloperoxidase gene. In addition to providing a potential route to the sustainable biological production of bromoform at scale, this work advances the development of novel microbial biosynthetic pathways for halogenation.

Considerable attention is given to absolute nutrient levels in lakes, rivers, and oceans, but less is paid to their relative concentrations, their nitrogen:phosphorus (N:P) stoichiometry, and the consequences of imbalanced stoichiometry. Here, we report 38 y of nutrient dynamics in Flathead Lake, a large oligotrophic lake in Montana, and its inflows. While nutrient levels were low, the lake had sustained high total N: total P ratios (TN:TP: 60 to 90:1 molar) throughout the observation period. N and P loading to the lake as well as loading N:P ratios varied considerably among years but showed no systematic long-term trend. Surprisingly, TN:TP ratios in river inflows were consistently lower than in the lake, suggesting that forms of P in riverine loading are removed preferentially to N. In-lake processes, such as differential sedimentation of P relative to N or accumulation of fixed N in excess of denitrification, likely also operate to maintain the lake's high TN:TP ratios. Regardless of causes, the lake's stoichiometric imbalance is manifested in P limitation of phytoplankton growth during early and midsummer, resulting in high C:P and N:P ratios in suspended particulate matter that propagate P limitation to zooplankton. Finally, the lake's imbalanced N:P stoichiometry appears to raise the potential for aerobic methane production via metabolism of phosphonate compounds by P-limited microbes. These data highlight the importance of not only absolute N and P levels in aquatic ecosystems, but also their stoichiometric balance, and they call attention to potential management implications of high N:P ratios.

The anaerobic digestion efficiency and methane production of straw was limited by its complex composition and structure. In this study, rice straw (RS), cellulose, and hemicellulose were used as raw materials to study biogas production performance and changes in the volatile fatty acids (VFAs). Further, microbial communities and genetic functions were analyzed separately for each material. The biogas production potential of RS, cellulose, and hemicellulose was different, with cumulative biogas production of 283.75, 412.50, and 620.64 mL/(g·VS), respectively. The methane content of the biogas produced from cellulose and hemicellulose was approximately 10% higher than that produced from RS after the methane content stabilized. The accumulation of VFAs occurred in the early stage of anaerobic digestion in all materials, and the cumulative amount of VFAs in both cellulose and hemicellulose was relatively higher than that in RS, and the accumulation time was 12 and 14 days longer, respectively. When anaerobic digestion progressed to a stable stage, Clostridium was the dominant bacterial genus in all three anaerobic digestion systems, and the abundance of Ruminofilibacter was higher during anaerobic digestion of RS. Genetically, anaerobic digestion of all raw materials proceeded mainly via aceticlastic methanogenesis, with similar functional components. The different performance of anaerobic digestion of RS, cellulose, and hemicellulose mainly comes from the difference of composition of raw materials. Increasing the accessibility of cellulose and hemicellulose in RS feedstock by pretreatment is an effective way to improve the efficiency of anaerobic digestion. Since the similar microbial community structure will be acclimated during anaerobic digestion, there is no need to adjust the initial inoculum when the accessibility of cellulose and hemicellulose changes., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The formation of dense, well-settling methanogenic granules is essential for the operation of high-rate, up-flow anaerobic bioreactors used for wastewater treatment. Granule formation (granulation) mechanisms have been previously proposed, but an ecological understanding of granule formation is still lacking. Additionally, much of the current research on granulation only examines the start-up phase of bioreactor operation, rather than monitoring the fate of established granules and how new granules emerge over time. This paper, therefore, attempts to provide an insight into the microbial ecology of granule formation outside the start-up phase of bioreactor operation and develop an ecological granulation model. The microbial communities of granules actively undergoing growth, breakage, and reformation were examined, and an ecological granulation model was proposed. A distinct pregranular microbial community, with a high proportion of acidogenic organisms, such as the Streptococcaceae , was identified and suggested to have a role in initiating granulation by providing simpler substrates for the methanogenic and syntrophic communities which developed during granule growth. After initial granule formation, deterministic influences on microbial community assembly increased with granule size and indicated that microbial community succession was influenced by granule growth, leading to the formation of a stepwise ecological model for granulation. IMPORTANCE Complex microbial communities in engineered environments can aggregate to form surface-attached biofilms. Others form suspended biofilms, such as methanogenic granules. The formation of dense, methanogenic granules underpins the performance of high-rate, anaerobic bioreactors in industrial wastewater treatment. Granule formation (granulation) has been well studied from a physico-chemical perspective, but the ecological basis is poorly understood. We identified a distinct, flocculent, microbial community, which was present alongside granules, comprising primary consumers likely key in providing simpler substrates to granules. This flocculent community is understudied in anaerobic digestion and may initiate, or perpetuate, granule formation. We propose that it may be possible to influence bioreactor performance (e.g., to regulate volatile fatty acid concentrations) by manipulating this community. The patterns of microbial community diversity and assembly revealed by the study indicate that cycles of granule growth and breakage lead to overall diversification of the bioreactor meta-community, with implications for bioreactor process stability.

This study investigated acid splitting wastewater (ASW) and interphase (IF) from soapstock splitting, as well as matter organic non glycerol (MONG) from glycerol processing, as potential substrates for biogas production. Batch and semicontinuous thermophilic anaerobic digestion experiments were conducted, and the substrates were preliminary treated using commercial enzymes kindly delivered by Novozymes A/C. The greatest enhancement in the batch digestion efficiency was achieved when three preparations; EversaTransform, NovoShape, and Lecitase were applied in the hydrolysis stage, which resulted in the maximum methane yields of 937 NL/kg VS and 915 NL/kg VS obtained from IF and MONG, respectively. The co-digestion of 68% ASW, 16% IF, and 16% MONG (wet weight basis) performed at an organic loading rate (OLR) of 1.5 kg VS/m 3 /day provided an average methane yield of 515 NLCH 4 /kg VS added and a volatile solid reduction of nearly 95%. A relatively high concentration of sulfates in the feed did not significantly affect the digestion performance but resulted in an increased hydrogen sulfide concentration in the biogas with the peak of 4000 ppm.

Wine production annually generates an estimated 11 million metric tonnes of grape marc (GM) worldwide. The diversion of this organic waste away from landfill and towards its use in the generation of renewable energy has been investigated. This study aimed to evaluate the effectiveness of operational parameters relating to the treatment regime and inoculum source in the extraction of methane from GM under unmixed anaerobic conditions at 35 °C. The study entailed the recirculation of a previously acclimated sludge (120 days) as downstream inoculum, an increased loading volume (1.3 kg) and a low substrate-to-inoculum ratio (10:3 SIR). The results showed that an incorporation of accessible operational controls can effectively enhance cumulative methane yield (0.145 m 3 CH 4 kg -1 VS), corresponding to higher amounts of digestible organics converted. The calculated average volumetric methane productivity equalled 0.8802 L CH 4 L Work -1 d -1 over 33.6 days whilst moderate pollutant removal (43.50% COD removal efficiency) was achieved. Molecular analyses identified Firmicutes and Bacteroidetes phyla as core organisms for hydrolytic and fermentative stages in trophic relationships with terminal electron acceptors from the methane-producing Methanosarcina genus. Economic projections established that the cost-effective operational enhancements were sustainable for valorisation from grape marc by existing wineries and distilleries.

Enteric fermentation from ruminants is a primary source of anthropogenic methane emission. This study aims to add another approach for methane mitigation by manipulation of the rumen microbiome. Effects of choline supplementation on methane formation were quantified in vitro using the Rumen Simulation Technique. Supplementing 200 mM of choline chloride or choline bicarbonate reduced methane emissions by 97-100% after 15 days. Associated with the reduction of methane formation, metabolomics analysis revealed high post-treatment concentrations of ethanol, which likely served as a major hydrogen sink. Metagenome sequencing showed that the methanogen community was almost entirely lost, and choline-utilizing bacteria that can produce either lactate, ethanol or formate as hydrogen sinks were enriched. The taxa most strongly associated with methane mitigation were Megasphaera elsdenii and Denitrobacterium detoxificans, both capable of consuming lactate, which is an intermediate product and hydrogen sink. Accordingly, choline metabolism promoted the capability of bacteria to utilize alternative hydrogen sinks leading to a decline of hydrogen as a substrate for methane formation. However, fermentation of fibre and total organic matter could not be fully maintained with choline supplementation, while amino acid deamination and ethanolamine catabolism produced excessive ammonia, which would reduce feed efficiency and adversely affect live animal performance., (© 2021. The Author(s).)

An estimated 25 million tons of animal manure is produced globally every year, causing considerable impact to the environment. These impacts can be managed through the use of anaerobic digestion (AD) This process achieves waste degradation through enzymatic activity, the efficiency of the AD process is directly related to microorganisms that produce these enzymes. Biomethane potential (BMP) assays remain the standard theoretical framework to pre-determine biogas yield and have been used to determine the feasibility of substrates or their combination for biogas production. However, an integrated approach that combines substrate choice and co-digestion would provide an improvement to the current predictive models. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) addresses the limitations of assays in this regard. In this paper, the biochemical functions of horse, cow, and pig manures are predicted. A total of 135 predicted KEGG Orthologies (KOs) showed amino acids, carbohydrate, energy, lipid, and xenobiotic metabolisms in all the samples. Linear discriminant analysis (LDA) combined with the effect size measurements (LEfSe), showed that fructose, mannose, amino acid and nucleotide sugar, phosphotransferase (PST) as well as starch and sucrose metabolisms were significantly higher in horse manure samples. 36 of the KOs were related to the acidogenesis and/or acetogenesis AD stages. Extended bar plots showed that 11 significant predictions were observed for horse-cow, while 5 were predicted for horse-pig and for cow-pig manures. Based on these predictions, the AD process can be enhanced through co-digestion strategies that takes into account the predicted metabolic contributions of the manure samples. The results supported the BMP calculations for the samples in this study. Biogas yields can be improved if this combined approach is employed in routine analysis before co-digesting different substrates., (© 2021. The Author(s).)

Aim: Spinal cord injury (SCI) patients may have intestinal dysmotility and digestive symptoms that are associated with small intestinal bacterial overgrowth (SIBO). The aim of this study is to describe the prevalence of SIBO in SCI patients and the risk factors of its development., Methods: Twenty-nine consecutive SCI patients were studied (10 women/19 men; mean age 47 years), 16 with subacute injuries (<9 months) and 13 with chronic injuries (>1 year). Nine patients were affected by tetraplegia and 15 by paraplegia. Each patient underwent a glucose breath test according to the North American Consensus and the presence of abdominal symptoms was evaluated during the test. The results were compared with 15 non-neurological patients with SIBO., Results: Six patients tested positive for SIBO (21%), all of them affected by SCI in the subacute phase, 6/16 vs. 0/13 in the chronic phase (P<.05) and the majority with tetraplegia, 5/9 vs. 1/19 with paraplegia (P<.05). No statistically significant relationship was found with other clinical characteristics. All the tests were positive for methane or mixed (methane and hydrogen), while only 67% of the controls had methane-predominant production (P>.05)., Conclusion: SCI patients can develop SIBO, more frequently in the subacute phase and in tetraplegic patients, highlighting a high production of methane. This complication should be considered in neurogenic bowel management., (Copyright © 2021 Elsevier España, S.L.U. All rights reserved.)

Background: Lactating dairy cows are the greatest livestock contributor of methane, a major global greenhouse gas (GHG). However, good feeding management with adequate mineral intake can offers an effective approach to maintaining high levels of milk production and the health of dairy cows over the entire course of lactation, while also helping to reduce methane emission. The study described here investigated the plasma concentrations of both macroelements (Ca, Na, K, Mg, P) and microelements (Zn, Cu, Fe, Mn), as well as enteric methane emission and milk composition in high-yielding dairy cows in different lactation periods. The experiment was performed on Holstein-Friesian dairy cows with the average milk yield of 41 (± 9) L/day in a Polish commercial farm with modern dairy systems. A total of thirty high-yielding dairy cows were randomly assigned into three groups differing by lactation stage: early stage (Early, days 25-100), middle stage (Middle, days 101-250), and late stage (Late, day 250 and later). Dietary treatment for all cows was a total mixture ration (TMR) with maize and alfalfa silage the main forage components., Results: The greatest milk yield and methane production were recorded in early-stage lactating cows, but the greatest methane intensity per kg of corrected milk was recorded in the late stage of lactation. Plasma concentrations of macroelements and microelements did not differ by lactation stages, but increased plasma concentrations of Zn and Fe and decreased plasma levels of Mg were noted during lactation. A positive correlation was found between plasma levels of Mg and other macroelements (Ca, Na, K), and between the concentrations of Fe and Zn, P in plasma, but no correlation between methane emission and mineral status was detected in the different lactation stages., Conclusions: Our results showed different mineral requirements and enteric methane emissions in each lactation stage. The feeding strategy and mineral utilization were adequate to maintain the health, mineral status, and milk production of the Holstein cows during the entire lactation period, and suggest an effective way of reducing methane emission., (© 2021. The Author(s).)

Foam accumulation in swine manure deep-pits has been linked to explosions and flash fires that pose devastating threats to humans and livestock. It is clear that methane accumulation within these pits is the fuel for the fire; it is not understood what microbial drivers cause the accumulation and stabilization of methane. Here, we conducted a 13-month field study to survey the physical, chemical, and biological changes of pit-manure across 46 farms in Iowa. Our results showed that an increased methane production rate was associated with less digestible feed ingredients, suggesting that diet influences the storage pit's microbiome. Targeted sequencing of the bacterial 16S rRNA and archaeal mcrA genes was used to identify microbial communities' role and influence. We found that microbial communities in foaming and non-foaming manure were significantly different, and that the bacterial communities of foaming manure were more stable than those of non-foaming manure. Foaming manure methanogen communities were enriched with uncharacterized methanogens whose presence strongly correlated with high methane production rates. We also observed strong correlations between feed ration, manure characteristics, and the relative abundance of specific taxa, suggesting that manure foaming is linked to microbial community assemblage driven by efficient free long-chain fatty acid degradation by hydrogenotrophic methanogenesis., Competing Interests: The authors have declared that no competing interests exist.

The basal zone of glaciers is characterized by physicochemical properties that are distinct from firnified ice due to strong interactions with underlying substrate and bedrock. Basal ice (BI) ecology and the roles that the microbiota play in biogeochemical cycling, weathering, and proglacial soil formation remain poorly described. We report on basal ice geochemistry, bacterial diversity (16S rRNA gene phylogeny), and inferred ecological roles at three temperate Icelandic glaciers. We sampled three physically distinct basal ice facies (stratified, dispersed, and debris bands) and found facies dependent on biological similarities and differences; basal ice character is therefore an important sampling consideration in future studies. Based on a high abundance of silicates and Fe-containing minerals and, compared to earlier BI literature, total C was detected that could sustain the basal ice ecosystem. It was hypothesized that C-fixing chemolithotrophic bacteria, especially Fe-oxidisers and hydrogenotrophs, mutualistically support associated heterotrophic communities. Basal ice-derived rRNA gene sequences corresponding to genera known to harbor hydrogenotrophic methanogens suggest that silicate comminution-derived hydrogen can also be utilized for methanogenesis. PICRUSt-predicted metabolism suggests that methane metabolism and C-fixation pathways could be highly relevant in BI, indicating the importance of these metabolic routes. The nutrients and microbial communities release from melting basal ice may play an important role in promoting pioneering communities establishment and soil development in deglaciating forelands., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Pretreatment and codigestion are proven to be effective strategies for the enhancement of the anaerobic digestion of lignocellulosic residues. The purpose of this study is to evaluate the effects of pretreatment and codigestion on methane production and the hydrolysis rate in the anaerobic digestion of agricultural wastes (AWs). Thermal and different thermochemical pretreatments were applied on AWs. Sewage sludge (SS) was selected as a cosubstrate. Biochemical methane potential tests were performed by mixing SS with raw and pretreated AWs at different mixing ratios. Hydrolysis rates were estimated by the best fit obtained with the first-order kinetic model. As a result of the experimental and kinetic studies, the best strategy was determined to be thermochemical pretreatment with sodium hydroxide (NaOH). This strategy resulted in a maximum enhancement in the anaerobic digestion of AWs, a 56% increase in methane production, an 81.90% increase in the hydrolysis rate and a 79.63% decrease in the technical digestion time compared to raw AWs. On the other hand, anaerobic codigestion (AcoD) with SS was determined to be ineffective when it came to the enhancement of methane production and the hydrolysis rate. The most suitable mixing ratio was determined to be 80:20 (Aws/SS) for the AcoD of the studied AWs with SS in order to obtain the highest possible methane production without any antagonistic effect.

Reports of biogenic methane (CH 4 ) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH 4 supersaturation of oxic surface waters has been termed the "methane paradox" because biological CH 4 synthesis is viewed to be a strictly anaerobic process carried out by O 2 -sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH 4 and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH 4 , suggesting that O 2 -insensitive, ecologically relevant aerobic CH 4 synthesis is likely of widespread distribution in the environment and should be considered in CH 4 modeling efforts., Competing Interests: The authors declare no competing interest.

Northern peatlands are experiencing more frequent and severe fire events as a result of changing climate conditions. Recent studies show that such a fire-regime change imposes a direct climate-warming impact by emitting large amounts of carbon into the atmosphere. However, the fires also convert parts of the burnt biomass into pyrogenic carbon. Here, we show a potential climate-cooling impact induced by fire-derived pyrogenic carbon in laboratory incubations. We found that the accumulation of pyrogenic carbon reduced post-fire methane production from warm (32 °C) incubated peatland soils by 13-24%. The redox-cycling, capacitive, and conductive electron transfer mechanisms in pyrogenic carbon functioned as an electron snorkel, which facilitated extracellular electron transfer and stimulated soil alternative microbial respiration to suppress methane production. Our results highlight an important, but overlooked, function of pyrogenic carbon in neutralizing forest fire emissions and call for its consideration in the global carbon budget estimation.