Dry Fermentation Research Articles

Simulation of livestock biomass resource recycling and energy utilization model based on dry type methane fermentation system

This study was aimed at investigating the local livestock biomass volume for dairy farms in Town A, located in eastern Hokkaido, Japan, and at presenting a model of a biogas plant that enables maximizing the use of the available livestock biomass. Using a dairy farm with 250 animals in Town A as a model for a biogas plant based on dry-type methane fermentation system (dry-type biogas plant), we set the operational conditions to an average hydraulic retention time of 20 days, digestion temperature of 55°C, and methane gas yield of 0.12 Nm3 CH4/kg VSA. We compared the biogas production of our presented model with that of a wet-type biogas plant with the same number of animals. The results showed that the dry-type biogas plant produced biogas at 859 Nm3/day, while the wet-type biogas plant produced biogas at 666 Nm3/day. These results indicate that introducing dry-type biogas plants in all dairy farms in Town A would potentially enable semi-solid livestock manure to be processed, which is not amenable to ordinary composting, in addition to the conventional processes being carried out through biogas plants, as well as lead to an increase in the amount of biogas production.

High-rate carboxylate production in dry fermentation of food waste at room temperature

Dry fermentation of food waste was optimized to achieve the maximum solid loading rate for carboxylate production without clogging events in a dry fermenter run at neutral pH. High inoculum-to-substrate ratio improved food waste solubilization and carboxylate yield, but the ratio 15% completely clogged the dry fermenter. Higher leachate circulate rate tended to enhance food waste fermentation, but partial clogging was observed at 13.2 L/h of leachate circulation rate. The dry fermenter achieved carboxylate yield of 428.5 g/kg food waste and volatile solid reduction of 79% at the solid loading rate 4.82 kg volatile solids/m3-d. This study first tracks chemical oxygen demand (COD) in food waste dry fermentation, showing maximum soluble COD <60% of food waste COD with residual food waste 13.6–16.3%. The operating cost was as low as $1.7/ton FW, implying that food waste treatment will be cost-neutral if recovered carboxylate can create economic benefit over the operating cost.

Effects of Different Pre-processing Methods on Dry Anaerobic Fermentation of Sugarcane Leaves and Pig Manure

Using agricultural wastes for anaerobic fermentation to produce biogas can not only realize the resource utilization of the wastes, but also prevent the environmental issues caused by straw burning. Sugarcane leaves contain waxy layer, which will cause problems such as difficulty in degradation, long-time fermentation and low biogas production. This paper studies the effects on three pre-processing methods of adding rush rot agent, natural retting for 7 days and water moisture for 24 hours of dry anaerobic fermentation of sugarcane leaves and pig manure. The results show that natural retting for 7 days has the advantage of daily biogas production when the fermentation period is less than 20 days, but this method is not obviously different from that of water moisture for 24 hours. When taking total biogas production volume and volume loading rate as the indicators, water moisture for 24 hours is the best pre-processing method for dry anaerobic fermentation of sugarcane leaves and pig manure when the fermentation period is more than 20 days.

Chemical and sensory perception of robusta coffees under wet processing

The quality of the coffee is determined by the sensorial analysis, using subjective methods and it is known that this quality is related to the chemical constituents of the grains. The present study used infrared spectroscopy to correlate the chemical composition of the grain with the final quality of the product and to assist in the current evaluation method, generating more reliability in the process. This study applied the spontaneous and induced fermentationwith starter cultures (yeast) to evaluate the sensory potential from the impacts generated by the types of processes adopted. The experiments were conducted in a randomized block design with five replicates, in a 3x4 factorial scheme, with three fermentation times: 24, 48, and 72 hours and fourwet processes: Washed, Yeast fermentation, Fully washed without yeast, and Fully washed with yeast. Infrared spectra were taken in the medium region(FTIR-ATR) of the 34 samples generated by the treatments. Given that the method of determining the quality of coffee is subjective, based on scores given by cuppers, the FTIR-ATR spectra were used in the construction of chemometric models to predict sensory ratings given to the attribute ‘acidity’. Themean error of prediction of the model constructed for the sensory score was 0.11. Sensory results indicated a higher score for the acidity attribute, in dry fermentation with yeast at the time of 48 hours, suggesting a new strategy for the production of quality robusta coffee.Key words: Fourier transform infrared spectroscopy; chemometrics; fermentation; acidity.

Coffee protein profiles during fermentation using different yeast inoculation methods

Abstract: The objective of this work was to evaluate the protein profiles of natural and semidry fermented Coffea arabica, either subjected to treatments with different yeast inoculation methods with starter culture or to an uninoculated control. Saccharomyces cerevisiae CCMA 0543 and Candida parapsilosis CCMA 0544 were separately inoculated into coffee by directly spraying the cherries on a terrace or in buckets, for 16 hours before sun drying. Protein quantification showed a significant difference between the protein profiles of the samples collected after natural dry fermentation. The MALDI-TOF MS analysis generated a list of 96 peaks with different mass-to-charge ratios (m/z) in the samples collected at the beginning and the end of fermentation. The highest number of peaks in the natural dry coffee was observed at the end of fermentation in the samples inoculated with S. cerevisiae CCMA 0543, in bucket, and in C. parapsilosis CCMA 0544 sprayed on the terrace. However, in the semidry processed coffee, the highest number of peaks was observed in the initial fermentation, with a decrease in the peptide peaks after fermentation. The fermentation with different microorganisms, processing types, and inoculation methods affects m/z profiles, influencing the types of proteins found in coffee.

Techno-economic assessment of dry fermentation in household biogas units through co-digestion of manure and agricultural crop residues in Egypt

In rural Egypt, communities face multiple challenges such as insufficient infrastructures for waste treatment, limited access to cheap energy and poor soils fertility. A decentralized, low-tech biogas technology for combined waste treatment and energy production, anaerobic digestion can achieve many sustainable development goals and resolve many issues faced by rural communities, treating and stabilizing organic waste into high-quality biofertilizer. Household biogas units for rural communities, however, should be subjected to techno-economic assessment to confirm their feasibility and technical efficiency. Therefore, this paper conducted: (1) survey of household biogas units and agricultural crop residues in rural communities in Egypt, (2) technical study of household biogas units, and (3) financial economic study. The results can be summarized as follows: (1) complete set of technical data on biogas units were documented, (2) crop residues were mapped using GIS software, (3) financial feasibility results were discussed. The total revenues were 1716.01, 2574.02, 3432.02, and 5148.04 EUR for the household biogas unit with the capacity of 2, 3, 4, and 6 m3, respectively. It was concluded that the units available in rural Egypt are considered as profitable projects from the profitability perspectives, and the values of the profitability indicators increased as the unit size increased.

PSVI-42 Effect of increasing concentrations of aflatoxins (AFB1) on rumen gas production (GP), dry matter (DM) disappearance and fermentation parameters using the in vitro ANKOM gas technique

Abstract Aflatoxins are secondary metabolites produced by Aspergillus species known to be the most prevalent contaminants in feedstuffs. In ruminants, contaminated AFB1 feeds usually exhibit symptoms including reduced feed efficiency and milk production and decreased appetite. The objective of this study was to investigate the effects of different concentrations of AFB1 on rumen fermentation parameters by using the ANKOM gas production protocol. Rumen fluid was collected from a cannulated dry dairy cow, filtered with cheese-cloth and diluted (1:1) with a standard buffer. Triplicates of 75 mL flasks were fed 0,75g of feed (79% corn silage, 15% alfalfa and 6% concentrates) and inoculated with 0 (blank), 0,2, 0,5, 1 and 2 ppm of AFB1. Flasks were placed in a rotating incubation at 39°C for 96h and connected to ANKOM GP system. After 96h of incubation, the contents of each flask were centrifuged. Supernatants were analyzed for total VFA and AFB1 while precipitates were dried at 104°C for DM disappearance. The experimentation was repeated weekly 3 times and named wk1, 2 and 3. Statistical analysis was done by SPSS using a univariate model. Results showed no significant differences on GP max at 96h among AFB1 concentrations. Only wk 1 showed that higher AFB1 concentration (2 ppm) decreased significantly (P &amp;lt; 0,05) DM disappearance (- 8,2 pts) when compared to the blank. Total VFA contents (75,0 ± 1,6 mM) were not affected by AFB1. Wk 2 and 3 did not show any difference neither on DM disappearance nor on VFA (89,1 ± 1,6 mM; 110,2 ± 4,8 mM). It can be concluded that our in vitro model, GP did not reflect DM disappearance and it can be put forward that rumen fluid with low total VFA concentrations (≤ 75 mM) could be more sensible to AFB1 challenge.

Enhanced nutritional value of chickpea protein concentrate by dry separation and solid state fermentation

Abstract A sustainable dry processing method to obtain nutritional and functional chickpea products was developed, yielding protein concentrates suited to prepare products without additives. Chickpeas were milled and air-classified into protein and starch-enriched concentrates. Subsequently, spontaneous solid state fermentation (SSF) with daily back-slopping was performed at 37 °C. The dominant autochthonous lactic acid bacteria (LAB) strains in chickpea flour and enriched fractions were Pediococcus pentosaceus and Pediococcus acidilactici. Strains were selected on their ability to metabolise flatulence-causing α-galactosides. SSF reduced the pH of the doughs in 24h from 6.6 to 4.2. After 72 h, concentrations of raffinose and stachyose were reduced by 88.3–99.1%, while verbascose became undetectable. Moreover, phytic acid reduced with 17% while total phenolic contents increased with 119%. Besides the observed differences in smell, texture and colour, the sourdoughs showed 67% higher water-holding capacity. This natural route to produce chickpea concentrates thus increases both the nutritive and technical functionality. Industrial relevance Increasing the sustainability of our food production is required to meet the demand for food of our growing world population. A processing route combining dry fractionation and solid state fermentation is developed to prepare chickpea concentrates with improved nutritional properties. This route is more sustainable as the use of water and thus energy-intensive drying steps are minimised. Moreover, it provides more natural ‘clean-label’ foods, i.e., foods with less additives and minimally processed. Solid state fermentation is used to reduce the presence of anti-nutritional factors, i.e., α-galactosides and phytic acid. Autochthonous LAB were accumulated via back-slopping and employed as starter culture as an alternative to the use of a commercial starter culture. Chickpea sourdoughs with enhanced nutritional quality of chickpea were obtained. The sourdough may be used directly to fortify cereal products like chickpea protein enriched bread or can be dried into a chickpea ingredient for many other applications as well.

Effect of fermentation and dry roasting on the nutritional quality and sensory attributes of quinoa.

BackgroundQuinoa is a pseudocereal with relatively high content of proteins and minerals that also contains mineral inhibitors such as phytate. The aim of the present study was to evaluate lactic acid fermentation and dry roasting on the nutritional quality and sensory attributes of quinoa. Various processes were evaluated, and quinoa grains were dry‐roasted, milled, and fermented, either with or without the addition of wheat phytase or activated quinoa phytase (added as back‐slop starter), for 10 hr. In other processes, raw quinoa flour was fermented for 10 hr or 4 hr and dry‐roasted. Hedonic sensory evaluation was then performed to evaluate the acceptability of the fermented flours prepared as porridges.ResultsThe combined dry roasting and fermentation processes significantly (p < .05) degraded phytate between 30% and 73% from initial content. The most effective process was fermentation of raw quinoa flour followed by dry roasting, which improved the estimated zinc and iron bioavailability. Particularly, estimated zinc bioavailability improved from low (Phy:Zn 25.4, Phy·Zn:Ca 295) to moderate (Phy:Zn 7.14, Phy·Zn:Ca 81.5). Phytate degradation was mainly attributed to the activation of endogenous phytase during fermentation. Dry roasting was effective in improving the sensory attributes of the fermented quinoa flour. Porridge made with raw quinoa flour fermented for 4 hr and dry‐roasted was more favorable to overall acceptability than that which was fermented for 10 hr and dry‐roasted.ConclusionFermentation of quinoa flour for 4 hr followed by dry roasting was successful in improving both nutritional and sensory attributes of the final product.

Control of Aflatoxin Production in Cassava Produced by Dry Fermentation in North Kivu, Democratic Republic of Congo

Traditionally, cassava (Manihot esculenta Crantz) is transformed by fermentation in water (retting) or in the open air (dry fermentation) in the DRC. In the east of the country (North Kivu), dry fermentation is the main technique for processing cassava for its detoxification and conservation. The Congolese farmers ferment the cassava to the open air using a preselected microferment contained in the scrapings of the fermented cassava previously called "MUSIYIRO". These fermentations are spontaneously directed by the microorganisms of the uncontrolled autochthonous flora. Unfortunately, toxinogenic molds are often more active in the fermentation process during which they also produce aflatoxins. This study was undertaken to help prevent the production of aflatoxins in cassava during this process. To do this, we substituted the traditional ferment with a strain of Rhizopus oryzae used as starter (microferment). Six successive replications, in controlled fermentation and uncontrolled fermentation, in a peasant environment (Beni, North Kivu) and fermentation directed by the strain of R. oryzae were carried out. Aflatoxins were then dosed in both groups of cassava flours. The results of the assay revealed an absence of aflatoxins in cassava fermented by scrapings from fermentation led by R. oryzea, while the non-directed fermentation controls were all contaminated with aflatoxins. These results show that it is possible to prevent the production of aflatoxins in cassava during fermentation when an aflatoxin-inhibiting microbial biomass is used which can progressively invade and colonize the fermentation site and thereby control the fermentation activities of cassava.

Screening of live yeast and yeast derivatives for their impact of strain and dose on in vitro ruminal fermentation and microbial profiles with varying media pH levels in high-forage beef cattle diet.

Yeast products showed beneficial effects with respect to stabilizing ruminal pH, stimulating ruminal fermentation and improving production efficiency. Batch cultures were conducted to evaluate the effects of yeast products on gas production (GP), dry matter disappearance (DMD) and fermentation characteristics of high-forage substrate. The study was a two media pH (5.8 and 6.5) × five yeasts (three live yeasts, LY: LY1, LY2, LY3; two yeast derivatives, YD: YD4, YD5) × four dosages factorial arrangement, with monensin (Mon) assigned as a positive control. Greater (P < 0.01) GP, DMD, volatile fatty acid (VFA) concentration, ratio of acetate to propionate (A:P) and copy numbers of Fibrobacter succinogenes and Ruminococcus flavefaciens were observed at pH6.5 than at pH5.8. The GP kinetics, DMD, VFA concentration, A:P and NH3 -N concentration differed (P < 0.05) among yeasts but varied with media pH or yeast dosages. Increasing doses of LY3 linearly increased DMD (P < 0.04) and VFA concentration (P < 0.001) at media pH5.8. The DMD linearly (P < 0.02) increased with increased addition of YD4 (pH6.5) and YD5 (pH5.8) and the ratio of A:P linearly decreased (P < 0.01) with the addition of YD4 or YD5 at pH5.8. Overall greater (P < 0.05) GP, A:P (pH5.8) and DMD (pH6.5) were observed with yeast products than with Mon. LY3 appeared to be an interesting candidate for improving rumen digestibility and fermentation efficiency, particularly at low media pH. YD4 or YD5 improved fermentation efficiency and can be potentially fed as an alternative to Mon. © 2019 Her Majesty the Queen in Right of Canada Journal of the Science of Food and Agriculture © 2019 Society of Chemical Industry.

Effects of dietary cation and anion difference on eating, ruminal function and plasma leptin in goats under tropical condition.

ObjectiveThis study was carried out to determine the effects of elevated dietary cation and anion difference (DCAD) on dry matter intake (DMI) and ruminal fermentation pattern in lactating dairy goats under tropical conditions.MethodsTen dairy goats were divided into two groups of five animals each. The groups received diets at different DCAD levels, either a control diet (22.81 mEq/100 g dry matter [DM], DCAD-23) or a DCAD-39 diet (39.08 mEq/100 g DM, DCAD-39). After parturition, DMI and water intake were recorded daily. Ruminal fluid and urine were collected, and nutrient digestibility measurements were carried out at 8th weeks postpartum (PP-8). Blood samples were collected at PP-4 and PP-8 to measure plasma leptin.ResultsDry matter intake/body weight (DMI/BW) at PP-8 of the animals fed the DCAD-39 diet was significantly higher than those fed with DCAD-23 diet (p<0.05). Animals fed with DCAD-39 consumed more water than those fed DCAD-23 over 24 h, particularly at night (p<0.05). Ruminal pH, acetate concentration, and urinary allantoin excretion increased with the DCAD-39 diet, whereas ruminal butyrate concentration was lower with the DCAD-39 diet. On the other hand, other ruminal parameters, such as total volatile fatty acid concentration, propionate molar proportion and acetate/propionate average ratio, were not affected by increased DCAD supplementation. Apparent digestibility was improved by increased DCAD supplementation. Plasma leptin concentration was higher with DCAD supplementation.ConclusionWhen feeding goats with DCAD-39 under tropical conditions, an increase in DMI was associated with improved apparent digestibility of nutrients, ruminal fermentation and microbial protein synthesis. An increase in plasma leptin concentration could not explain the effect of high DCAD on DMI.

Effects of size and autoclavation of fruit and vegetable wastes on biohydrogen production by dark dry anaerobic fermentation under mesophilic condition

Fermentative hydrogen production from fruit and vegetable wastes (FVWs) through Dry Fermentation Technology (DFT) was studied through three independent experiments in order to find out the effect of particle size and autoclaving pretreatment on bio-hydrogen production from FVWs and as follows: (1) autoclaved FVWs with sizes < 5 cm (experiment I); (2) raw FVWs with sizes < 5 cm (experiment II) and (3) autoclaved FVWs with sizes > 5 cm (experiment III). The assay with autoclaved waste yielded a higher percentage of hydrogen in the headspace of the dry fermenter reaching a maximum value of 44% in experiment I. However, the maximum hydrogen production was obtained in experiment III with 14573 NmL at a yield of 23.53 NmL H2/gVS. Profiling of the microbial communities by denaturing gradient gel electrophoresis (DGGE) indicated that the most prominent species were the genera Clostridium, Bifidobacterium, and Lactobacillus.

Influence of dietary probiotic inclusion on growth performance, nutrient utilization, ruminal fermentation activities and methane production in growing lambs

The use of two probiotic products as a means of improving in vitro and ruminal dry matter digestibility, fermentation characteristics and growth performance of Barki lambs were evaluated. The probiotic products in powder (PP) or liquid (PL) forms were produced from Ruminococcus flavefaciens, through an anaerobic fermentation process. Barki lambs (n = 30; 24.5 ± 0.5 kg body weight) were used in a completely randomized block design and randomly assigned among three experimental groups and fed for 75 days. Lambs were fed an equal amount of concentrate feed mixture with either no probiotic product (control), or with 20 g of PP, or 10 ml of PL per animal/d, with rice straw ad libitum. Both PP and PL treatments resulted in an increase (p < 0.05) in nearly all of the digestibility coefficients, nitrogen utilization, cell wall constituents, total volatile fatty acids, rumen volume, microbial nitrogen synthesis, gas production and average daily gain compared to the control group. Ruminal ammonia nitrogen, acetic acid and in vitro methane concentrations and protozoa count decreased (p < 0.03) in the treatment groups. Overall, the probiotic in a powder or liquid form increased in vitro dry matter digestibility, improved lambs daily gain and nutrient digestibility.

Advances in dry fermentation extends biowaste management possibilities

ABSTRACTIt was repeatedly and independently confirmed that processing of biowaste via dry fermentation technology is less capital demanding compared to a conventional (also known as continuous, wet and mixable) fermentation technology. However, low acquisition and running cost are redeemed by lower intensity of the fermentation process that results in small revenues. In this study, biowaste was deeply disintegrated before it entered the dry anaerobic fermentation that was followed by pyrolysis of the fermentation residues. It was revealed that the disintegration step increases the intensity of the fermentation process, therefore biogas yields are increased, hydraulic retention times could be shortened and biochar is priced higher (because of higher porosity partially caused during the fermentation process). Additional experiments on a commercial scale indicate that it is economically beneficial (reduction of the payback period by more than 7%) to perform the disintegration step later, in the middle phase of the fermentation process.

Influence Mechanism of Micro-Oxygenation Treatment on Dry Fermentation of Food Waste

Influence Mechanism of Micro-Oxygenation Treatment on Dry Fermentation of Food Waste

Effect of percolation frequency on biohydrogen production from fruit and vegetable wastes by dry fermentation

Organic solid wastes are the most abundant sources for biohydrogen production. Dry fermentation system has many advantages over continuously fed reactor systems for treatment of organic solid wastes. In this study the effect of percolation frequency on yield of biohydrogen production from fruit and vegetable wastes using dry fermentation system was examined. For this purpose 2 times percolation per day, 1 time percolation per day and 1 time percolation per 2 days frequency were compared and the hydrogen yields were observed as; 57 mL H2/gVSremoved, 53 mL H2/gVSremoved and 68 mL H2/gVSremoved respectively. The percolation frequency didn't affect the overall yield but significantly affected the biohydrogen producing reactor of the dry fermentation system. 80% of the hydrogen was produced in percolation tank during 1 time per 2 days feeding and almost all hydrogen production was conducted in dry fermenter during 2 times per day percolation. Therefore the percolation frequency is found to be very important for system operation characteristics.

Biohydrogen production from autoclaved fruit and vegetable wastes by dry fermentation under thermophilic condition

Dark fermentative hydrogen production from organic waste is an attractive technique that simultaneously treats waste along with generation of renewable fuel. In this study, a relative new technology named dark dry fermentation was tested in a 55-L reactor to treat fruit and vegetable waste (FVW) along with simultaneous generation of biohydrogen. To understand the effect of autoclaving as a pretreatment method on FVW for subsequent biohydrogen production, two independent experiments were performed; one with autoclaved waste (experiment I) and another by using non autoclaved waste (experiment II). From the analyses, it was found that maximum hydrogen % obtained for experiment I was 41% (v/v%) whereas, for experiment II was 21%. In terms of total hydrogen produced, around 30% higher production was observed with experiment I compared to experiment II. The hydrogen yields for experiment I and experiment II were respectively, 27.19 and 20.81 NmL H2/gVS (VS = volatile solid added), and the metabolites (VFAs) preferentially produced were acetic acid and iso-butyric acid.

Fermentative profile of maize silage inoculated with Lactobacillus buchneri

Biological silage additives can assist in making silages by promoting a rapid reduction in silage pH and preventing aerobic deterioration. The current Lactobacillus buchnerion the market produces acetic acid slowly and identifying strains that would improve aerobic stability earlier in the ensiling process would be helpful. This study aimed to investigate the changes in microbial population, dry matter (DM) recovery and fermentation profile of maize silage with or without inoculation with L. buchneri after 45 days of ensiling. The wild L. buchneri strains were isolated from tropical maize silage in a previous study. Four strains of L. buchneri (56.22, 56.27, 56.28 and 56.29) were used as inoculants. Data from the silo openings were analyzed as a completely randomized design, with four replicates per treatment (inoculants). Selected strains did not affect the DM content, yeast and mould population, DM recovery, water-soluble carbohydrates (WSC), lactic acid and butyric acid of maize silage after 45 days of ensiling (p> 0.05). The pH, lactic acid bacteria (LAB) population and concentrations of acetic and propionic acids and ethanol were affected by inoculants (p < 0.05). The strains 56.22, 56.27 and 56.28 showed lower pH than the untreated control silage, but lower acetic acid concentration

PSI-14 Humic substances alter ammonia production and the microbial populations within a RUSITEC fed a mixed hay – concentrate diet.

The aim of this study was to examine the effects of humic substances on fermentation characteristics and microbial communities using the rumen stimulation technique (RUSITEC). The experiment was conducted as a completely randomized design over a 15-d period with 3 treatments duplicated in 2 runs with 2 replicates per run. Treatments consisted of a control diet (forage:concentrate; 60:40) without humic substances or humic substances added at either 1.5 g/d or 3.0 g/d. Dry matter disappearance, pH, fermentation parameters and gas production were measured from d 8 to 15. Samples for microbial profiling were taken on d 5, 10 and 15 using the digested feed bags for solid-associated microbes (SAM) and fermenter fluid for liquid-associated microbes (LAM). The inclusion of humic substances had no effect (P ≥ 0.19) on DM disappearance, pH or the concentrations of VFA. The production of NH3 was linearly decreased (P = 0.04) with increasing levels of humic substances in the diet. There was no effect (P ≥ 0.43) of humic substances on total gas, CO2 or CH4 production. The number of OTUs was significantly reduced in the 3.0 g/d treatment compared to the control on d 10 and 15; however, the microbial community structure was largely unaffected (P > 0.05). In the SAM samples, the genera Lachnospiraceae XPB1014 group, Succiniclasticum, and Fibrobacter were reduced in the 3.0 g/d treatment and Anaeroplasma, Olsenella, and Pseudobutyrivibrio were increased at d 5, 10, or 15. Within the LAM samples, Christensenellaceae R-7 and Succiniclasticum were the most differentially abundant genera between the control and 3.0 g/d HS treatment samples (P < 0.05). This study highlights the potential use of humic substances as a natural feed additive which may play a role in nitrogen metabolism without negatively affecting the ruminal microbiota.