Bioaugmentation as a strategy for tailor-made volatile fatty acid production.

This study aimed to develop a novel strategy for tailor-made volatile fatty acid (VFA) composition. For this purpose, the mixed microbial culture was bioaugmented by Propionibacterium acidipropionici. Anaerobic sequencing batch reactors were operated with cheese wastewater under alkali pH. While the maximum propionic acid production almost four times increased (3779 ± 201 mgCODeq propionic acid/L in the bioaugmented reactor and 942 ± 172 mgCODeq propionic acid/L in the control reactor), there was no significant difference in VFA composition. The gene copy number of P.acidipropionici increased 20 times after the bioaugmentation. Furthermore, the gene copy number of P.acidipropionici was positively correlated with total VFA and isovaleric acid concentration. The relative abundance of family Flavobacteriaceae increased in the bioaugmented reactor, which might be caused by the syntrophic relation between Flavobacteriaceae and P. acidipropionici. The cycle analysis results showed that the shorter cycle (6h) could ensure the same efficiency.

Efficient co-production of propionic acid and succinic acid by Propionibacterium acidipropionici using membrane separation coupled technology.

To improve the fermentation efficiency of Propionibacterium acidipropionici, a semi-continuous coupled fermentation process was established to achieve co-production of propionic acid (PA) and succinic acid (SA). First, the optimal proportion of glucose (Glc) and glycerol (Gl) as a mixed carbon source was determined, and the feeding procedure of Gl was optimized to make more energy flow in the direction of product synthesis. Then, ZGD630 anion exchange resin was used for efficient adsorption of PA, thereby eliminating the feedback inhibition effect of PA. Finally, an efficient, coupled fermentation process of P. acidipropionici characterized by membrane separation and chromatography technology was developed. The concentrations of PA and SA reached 62.22 ± 2.32 and 20.45 ± 1.34 g L-1, with corresponding productivity of 0.43 and 0.14 g L-1 h-1, increased by 65.38% and 48.54%, respectively. Membrane separation coupled fermentation of PA and SA could significantly improve the process economics of P. acidipropionici, and has good prospects for industrial application.

Carbon nanotubes mitigate copper-oxide nanoparticles-induced inhibition to acidogenic metabolism of Propionibacterium acidipropionici by regulating carbon source utilization.

This study demonstrated that multi-walled carbon nanotubes (MWCNTs) could mitigate copper oxide nanoparticles (CuO NPs)-induced inhibition to acidogenic metabolism of propionic acid bacteria (i.e., Propionibacterium acidipropionici) by regulating carbon source utilization. CuO NPs severely inhibited the growth of P. acidipropionici, damaged its cell membrane, and down-regulated gene expressions and enzyme activities involved in acidogenic metabolism, thereby decreasing propionate production. However, although MWCNTs had a slightly negative impact on the growth and cell membrane, the gene expressions and catalytic activities were enhanced (glycolysis and pyruvate metabolism), resulting in the improved propionate production. Additionally, the gene expressions and catalytic activities of key enzymes (e.g., tpiA, pgk, PK, OTTAC, etc.) related to acidogenic metabolism were also enhanced by the co-existence of both nanomaterials, thereby promoting propionate production towards P. acidipropionici. This work demonstrated that the presence of MWCNTs could affect the inhibition of CuO NPs to fermentation processes via regulating carbon source utilization.

Improving propionic acid production from a hemicellulosic hydrolysate of sorghum bagasse by means of cell immobilization and sequential batch operation.

Propionic acid (PA) is an important organic compound with extensive application in different industrial sectors and is currently produced by petrochemical processes. The production of PA by large-scale fermentation processes presents a bottleneck, particularly due to low volumetric productivity. In this context, the present work aimed to produce PA by a biochemical route from a hemicellulosic hydrolysate of sorghum bagasse using the strain Propionibacterium acidipropionici CIP 53164. Conditions were optimized to increase volumetric productivity and process efficiency. Initially, in simple batch fermentation, a final concentration of PA of 17.5 g⋅L-1 was obtained. Next, fed batch operation with free cells was adopted to minimize substrate inhibition. Although a higher concentration of PA was achieved (38.0 g⋅L-1 ), the response variables (YP/S = 0.409 g⋅g-1 and QP = 0.198 g⋅L-1 ⋅H-1 ) were close to those of the simple batch experiment. Finally, the fermentability of the hemicellulosic hydrolysate was investigated in a sequential batch with immobilized cells. The PA concentration achieved a maximum of 35.3 g⋅L-1 in the third cycle; moreover, the volumetric productivity was almost sixfold higher (1.17 g⋅L-1 ⋅H-1 ) in sequential batch than in simple batch fermentation. The results are highly promising, providing preliminary data for studies on scaling up the production of this organic acid.

Transcriptomics and Proteomics Analyses of the Responses of Propionibacterium acidipropionici to Metabolic and Evolutionary Manipulation.

We first performed a combination of metabolic engineering (deletion of ldh and poxB and overexpression of mmc) with evolutionary engineering (selection under oxygen stress, acid stress and osmotic stress) in Propionibacterium acidipropionici. The results indicated that the mutants had superior physiological activity, especially the mutant III obtained from P. acidipropionici-Δldh-ΔpoxB+mmc by evolutionary engineering, with 1.5-3.5 times higher growth rates, as well as a 37.1% increase of propionic acid (PA) titer and a 37.8% increase PA productivity compared to the wild type. Moreover, the integrative transcriptomics and proteomics analyses revealed that the differentially expressed genes (DEGs) and proteins (DEPs) in the mutant III were involved in energy metabolism, including the glycolysis pathway and tricarboxylic acid cycle (TCA cycle). These genes were up-regulated to supply increased amounts of energy and precursors for PA synthesis compared to the wild type. In addition, the down-regulation of fatty acid biosynthesis and fatty acid metabolism may indicate that the repressed metabolic flux was related to the production of PA. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to verify the differential expression levels of 16 selected key genes. The results offer deep insights into the mechanism of high PA production, which provides the theoretical foundation for the construction of advanced microbial cell factories.

Propionic Acid: Method of Production, Current State and Perspectives.

During the past years, there has been a growing interest in the bioproduction of propionic acid by Propionibacterium. One of the major limitations of the existing models lies in their low productivity yield. Hence, many strategies have been proposed in order to circumvent this obstacle. This article provides a comprehensive synthesis and review of important biotechnological aspects of propionic acid production as a common ingredient in food and biotechnology industries. We first discuss some of the most important production processes, mainly focusing on biological production. Then, we provide a summary of important propionic acid producers, including Propionibacterium freudenreichii and Propionibacterium acidipropionici, as well as a wide range of reported growth/production media. Furthermore, we describe bioprocess variables that can have impact on the production yield. Finally, we propose methods for the extraction and analysis of propionic acid and put forward strategies for overcoming the limitations of competitive microbial production from the economical point of view. Several factors influence the propionic acid concentration and productivity such as culture conditions, type and bioreactor scale; however, the pH value and temperature are the most important ones. Given that there are many reports about propionic acid production from glucose, whey permeate, glycerol, lactic acid, hemicelluloses, hydrolyzed corn meal, lactose, sugarcane molasses and enzymatically hydrolyzed whole wheat flour, only few review articles evaluate biotechnological aspects, i.e. bioprocess variables.

Soybean whole-plant ensiled with chitosan and lactic acid bacteria: Microorganism counts, fermentative profile, and total losses.

Chitosan is a biopolymer derived from chitin deacetylation, present in the exoskeleton of crustaceans and insects. Chitosan has been evaluated as rumen modulator and silage additive due to its antimicrobial properties. The objective of this study was to determine the effects of both chitosan and a bacterial additive on microbiological quality, chemical composition, nutrient in vitro degradation, fermentative profile, and total losses of whole-soybean plant silage (SS) harvested at R6 stage. Four treatments in a factorial arrangement were randomly assigned to 40 experimental minisilos as no additives (CON), 8 g/t fresh forage of microbial inoculant (INO; Kera SIL, Kera Nutrição Animal, Bento Gonçalves, Brazil); 5 g/kg of fresh forage chitosan (CHI); and CHI + INO. Microbial inoculant was composed of Lactobacillus plantarum (4.0 × 1010 cfu/g) and Propionibacterium acidipropionici (2.6 × 1010 cfu/g). The CHI and INO alone increased counts of lactic bacteria and anaerobic bacteria and decreased counts of mold and yeast in SS. The CHI or INO alone increased in vitro degradation of dry matter, crude protein, and neutral detergent fiber, and decreased nonfiber carbohydrate content of SS. Chitosan increased NH3-N and lactate concentrations and decreased ethanol concentration in SS. The CHI increased dry matter recovery from SS; INO increased silage aerobic stability. The combination of CHI+INO showed the lowest value of gas losses. In general, the combination of CHI and INO had small positive effects on gas losses of SS; however, both CHI or INO alone improved nutrient in vitro degradation and decreased mold and yeast in SS. Chitosan or INO utilization improves SS quality.

Effect of re-ensiling on the quality of sorghum silage.

The commercialization of silage in many countries, including Brazil, has increased in recent years. Re-ensiling of previously ensiled forage occurs when silage is relocated from one farm to another, where it will be compacted and sealed again. During this process, silage is exposed to oxygen before being ensiled, which may affect its quality. We exposed sorghum silage to air during the anaerobic storage phase to simulate the transportation of silages between farms. Experimental treatments included silage exposed to air for 0 or 12 h, with or without the use of an inoculant containing a mixture of Lactobacillus plantarum and the propionic bacteria Propionibacterium acidipropionici (1 × 106 cfu/g of forage; Biomax corn, Lallemand, Saint-Simon, France), totaling 4 treatments: conventional silage, conventional silage with inoculant use, re-ensilage after exposure to air, and re-ensilage after exposure to air with use of an inoculant. The sorghum was stored in experimental silos containing about 9.0 kg of fresh forage per replicate. Treatments were tested in a factorial 2 × 2 design with 5 replicates each. Chemical composition, in vitro dry matter digestibility, fermentative characteristics, losses (due to gas, effluents, and total dry matter), microorganism counts, and aerobic stability of sorghum silage were evaluated. Dry matter content of sorghum before ensiling was 273.12 g/kg. The 12-h re-ensiling process increased the effluent loss of the silage when compared with conventional silage (456.42 vs. 201.19 g/kg of FM, respectively). In addition, re-ensiled silages presented lower concentrations of lactic acid and higher concentrations of propionic acid than the silages that had not been opened during storage. The aerobic stability of silage was not affected by the re-ensiling process and the use of inoculant. The use of inoculant increased the pH and loss of dry matter of the silages (4.23 vs. 3.98 and 14.05 vs. 7.82%, respectively) and therefore did not provide any benefits in this study.

Comparative genomics and transcriptomics analysis-guided metabolic engineering of Propionibacterium acidipropionici for improved propionic acid production.

Acid stress induced by the accumulation of organic acids during the fermentation of propionibacteria is a severe limitation in the microbial production of propionic acid (PA). To enhance the acid resistance of strains, the tolerance mechanisms of cells must first be understood. In this study, comparative genomic and transcriptomic analyses were conducted on wild-type and acid-tolerant Propionibacterium acidipropionici to reveal the microbial response of cells to acid stress during fermentation. Combined with the results of previous proteomic and metabolomic studies, several potential acid-resistance mechanisms of P. acidipropionici were analyzed. Energy metabolism and transporter activity of cells were regulated to maintain pH homeostasis by balancing transmembrane transport of protons and ions; redundant protons were eliminated by enhancing the metabolism of certain amino acids for a relatively stable intracellular microenvironment; and protective mechanism of macromolecules were also induced to repair damage to proteins and DNA by acids. Transcriptomic data indicated that the synthesis of acetate and lactate were undesirable in the acid-resistant mutant, the expression of which was 2.21-fold downregulated. In addition, metabolomic data suggested that the accumulation of lactic acid and acetic acid reduced the carbon flow to PA and led to a decrease in pH. On this basis, we propose a metabolic engineering strategy to regulate the synthesis of lactic acid and acetic acid that will reduce by-products significantly and increase the PA yield by 12.2% to 10.31 ± 0.84 g/g DCW. Results of this study provide valuable guidance to understand the response of bacteria to acid stress and to construct microbial cell factories to produce organic acids by combining systems biology technologies with synthetic biology tools.

Propionic acid production from soy molasses by Propionibacterium acidipropionici: Fermentation kinetics and economic analysis.

Propionic acid (PA) is a specialty chemical; its calcium salt is widely used as food preservative. Soy molasses (SM), a low-value byproduct from soybean refinery, contains sucrose and raffinose-family oligosaccharides (RFO), which are difficult to digest for most animals and industrial microorganisms. The feasibility of using SM for PA production by P. acidipropionici, which has genes encoding enzymes necessary for RFO hydrolysis, was studied. With corn steep liquor as the nitrogen source, stable long-term PA production from SM was demonstrated in sequential batch fermentations, achieving PA productivity of >0.8 g/L h and yield of 0.42 g/g sugar at pH 6.5. Economic analysis showed that calcium propionate as the main component (63.5%) in the product could be produced at US $1.55/kg for a 3000-MT plant with a capital investment of US $10.82 million. At $3.0/kg for the product, the process offers attractive 40% return of investment and is promising for commercial application.

Effect of Propionibacterium acidipropionici P169 on the rumen and faecal microbiota of beef cattle fed a maize-based finishing diet.

Direct fed microbial supplementation with lactic acid utilising bacteria (i.e. Propionibacterium acidipropionici P169) has been shown to alleviate the severity of subacute ruminal acidosis in high-grain fed beef cattle. This study was carried out to explore the impact of P169 supplementation on modulating rumen and hindgut microbiota of high-grain fed steers. Seven ruminally-canulated high-grain fed steers were randomly assigned to two treatment groups: control diet (n=3) and the same diet supplemented with P169 added at a rate of 1×1011 cfu/head/d (n=4). Samples were collected every 28 days for a 101 d period (5 time points) and subjected to qPCR quantification of P169 and high-throughput sequencing of bacterial V4 16S rRNA genes. Ruminal abundance of P169 was maintained at elevated levels (P=0.03) both in liquid and solid fractions post supplementation. Concomitant with decreased proportion of amylolytic (such as Prevotella) and key lactate-utilisers (such as Veillonellaceae and Megasphaera), the proportions of cellulolytic bacterial lineages (such as Ruminococcaceae, Lachnospiraceae, Clostridiaceae, and Christensenellaceae) were enriched in the rumen microbiota of P169-supplemented steers. These, coupled with elevated molar proportions of branched-chain fatty acids and increased concentration of ammonia in the rumen content of P169-supplemented steers, indicated an improved state of fibrolytic and proteolytic activity in response to P169 supplementation. Further, exploring the hindgut microbiota of P169-supplemented steers revealed enrichment of major amylolytic bacterial lineages, such as Prevotella, Blautia, and Succinivibrionaceae, which might be indicative of an increased availability of carbohydrates in the hindgut ecosystem following P169 supplementation. Collectively, the present study provides insights into the microbiota dynamics that underlie the P169-associated shifts in the rumen fermentation profile of high-grain fed steers.

Awakening sleeping beauty: production of propionic acid in Escherichia coli through the sbm operon requires the activity of a methylmalonyl-CoA epimerase.

BACKGROUND: Propionic acid is used primarily as a food preservative with smaller applications as a chemical building block for the production of many products including fabrics, cosmetics, drugs, and plastics. Biological production using propionibacteria would be competitive against chemical production through hydrocarboxylation of ethylene if native producers could be engineered to reach near-theoretical yield and good productivity. Unfortunately, engineering propionibacteria has proven very challenging. It has been suggested that activation of the sleeping beauty operon in Escherichia coli is sufficient to achieve propionic acid production. Optimising E. coli production should be much easier than engineering propionibacteria if tolerance issues can be addressed. RESULTS: Propionic acid is produced in E. coli via the sleeping beauty mutase operon under anaerobic conditions in rich medium via amino acid degradation. We observed that the sbm operon enhances amino acids degradation to propionic acid and allows E. coli to degrade isoleucine. However, we show here that the operon lacks an epimerase reaction that enables propionic acid production in minimal medium containing glucose as the sole carbon source. Production from glucose can be restored by engineering the system with a methylmalonyl-CoA epimerase from Propionibacterium acidipropionici (0.23 ± 0.02 mM). 1-Propanol production was also detected from the promiscuous activity of the native alcohol dehydrogenase (AdhE). We also show that aerobic conditions are favourable for propionic acid production. Finally, we increase titre 65 times using a combination of promoter engineering and process optimisation. CONCLUSIONS: The native sbm operon encodes an incomplete pathway. Production of propionic acid from glucose as sole carbon source is possible when the pathway is complemented with a methylmalonyl-CoA epimerase. Although propionic acid via the restored succinate dissimilation pathway is considered a fermentative process, the engineered pathway was shown to be functional under anaerobic and aerobic conditions.

Improved production of propionic acid using genome shuffling.

Traditionally derived from fossil fuels, biological production of propionic acid has recently gained interest. Propionibacterium species produce propionic acid as their main fermentation product. Production of other organic acids reduces propionic acid yield and productivity, pointing to by-products gene-knockout strategies as a logical solution to increase yield. However, removing by-product formation has seen limited success due to our inability to genetically engineer the best producing strains (i.e. Propionibacterium acidipropionici). To overcome this limitation, random mutagenesis continues to be the best path towards improving strains for biological propionic acid production. Recent advances in next generation sequencing opened new avenues to understand improved strains. In this work, we use genome shuffling on two wild type strains to generate a better propionic acid producing strain. Using next generation sequencing, we mapped the genomic changes leading to the improved phenotype. The best strain produced 25% more propionic acid than the wild type strain. Sequencing of the strains showed that genomic changes were restricted to single point mutations and gene duplications in well-conserved regions in the genomes. Such results confirm the involvement of gene conversion in genome shuffling as opposed to long genomic insertions.

Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for xylose fermentation.

Propionibacterium freudenreichii cannot use xylose, the second most abundant sugar in lignocellulosic biomass. Although Propionibacterium acidipropionici can use xylose as a carbon source, it is difficult to genetically modify, impeding further improvement through metabolic engineering. This study identified three xylose catabolic pathway genes encoding for xylose isomerase (xylA), xylose transporter (xylT), and xylulokinase (xylB) in P. acidipropionici and overexpressed them in P. freudenreichii subsp. shermanii via an expression plasmid pKHEM01, enabling the mutant to utilize xylose efficiently even in the presence of glucose without glucose-induced carbon catabolite repression. The mutant showed similar fermentation kinetics with glucose, xylose, and the mixture of glucose and xylose, respectively, as carbon source, and with or without the addition of antibiotic for selection pressure. The engineered P. shermanii thus can provide a novel cell factory for industrial production of propionic acid and other value-added products from lignocellulosic biomass.

Efficient production of propionic acid through high density culture with recycling cells of Propionibacterium acidipropionici.

The aim of this study was to explore propionic acid production via high density culture of Propionibacterium acidipropionici and recycling of cells. Results showed that final cells of P. acidipropionici from high density culture still had high metabolic activity for reuse. Using our process, 75.9gl(-1) propionic acid was produced, which was 1.84-fold of that in fed-batch fermentation with low cell density (41.2gl(-1)); the corresponding productivity was 100.0% higher than that in fed-batch fermentation with low cell density (0.16gl(-1)h(-1)). This bioprocess may have potential for the industrial production of propionic acid.

Microbial production of Propionic and Succinic acid from Sorbitol using Propionibacterium acidipropionici.

Three sequential fermentative batches were carried out with cell recycle in four simultaneously operating bioreactors maintained at pH 6.5, 30°C, and 100 rpm. P. acidipropionici ATCC 4875 was able to produce propionic and succinic acid from sorbitol. The concentration of propionic acid decreased slightly from 39.5 ± 5.2 g L(-1) to 34.4 ± 1.9 g L(-1), and that of succinic acid increased significantly from 6.1 ± 2.1 g L(-1) to 14.8 ± 0.9 g L(-1) through the sequential batches. In addition, a small amount of acetic acid was produced that decreased from 3.3 ± 0.4 g L(-1) to 2.0 ± 0.3 g L(-1) through the batches. The major yield for propionic acid was 0.613 g g(-1) in the first batch and succinic acid it was 0.212 g g(-1) in the third batch. The minor yield of acetic acid was 0.029 g g(-1), in the second and third batches.

Effects of carbon dioxide on cell growth and propionic acid production from glycerol and glucose by Propionibacterium acidipropionici.

The effects of CO2 on propionic acid production and cell growth in glycerol or glucose fermentation were investigated in this study. In glycerol fermentation, the volumetric productivity of propionic acid with CO2 supplementation reached 2.94g/L/day, compared to 1.56g/L/day without CO2. The cell growth using glycerol was also significantly enhanced with CO2. In addition, the yield and productivity of succinate, the main intermediate in Wood-Werkman cycle, increased 81% and 280%, respectively; consistent with the increased activities of pyruvate carboxylase and propionyl CoA transferase, two key enzymes in the Wood-Werkman cycle. However, in glucose fermentation CO2 had minimal effect on propionic acid production and cell growth. The carbon flux distributions using glycerol or glucose were also analyzed using a stoichiometric metabolic model. The calculated maintenance coefficient (mATP) increased 100%, which may explain the increase in the productivity of propionic acid in glycerol fermentation with CO2 supplement.

Improved propionic acid production from glycerol: combining cyclic batch- and sequential batch fermentations with optimal nutrient composition.

Propionic acid was produced from glycerol using Propionibacterium acidipropionici. In this study, the impact of the concentrations of carbon and nitrogen sources, and of different modes of high cell density fermentations on process kinetics and -efficiency was investigated. Three-way ANOVA analysis and batch cultivations at varying C/N ratios at pH 6.5 revealed that propionic acid production rate is significantly influenced by yeast extract concentration. Glycerol to yeast extract ratio (ww(-1)) of 3:1 was required for complete glycerol consumption, while maintaining the volumetric productivity. Using this optimum C/N ratio for propionic acid production in cyclic batch fermentation gave propionate yield up to 93mol% and productivity of 0.53gL(-1)h(-1). Moreover, sequential batch fermentation with cell recycling resulted in production rates exceeding 1gL(-1)h(-1) at initial glycerol up to 120gL(-1), and a maximum of 1.63gL(-1)h(-1) from 90gL(-1) glycerol.

High cell density propionic acid fermentation with an acid tolerant strain of Propionibacterium acidipropionici.

Propionic acid is an important chemical with wide applications and its production via fermentation is of great interest. However, economic production of bio-based propionic acid requires high product titer, yield, and productivity in the fermentation. A highly efficient and stable high cell density (HCD) fermentation process with cell recycle by centrifugation was developed for propionic acid production from glucose using an acid-tolerant strain of Propionibacterium acidipropionici, which had a higher specific growth rate, productivity, and acid tolerance compared to the wild type ATCC 4875. The sequential batch HCD fermentation at pH 6.5 produced propionic acid at a high titer of ∼40 g/L and productivity of 2.98 g/L h, with a yield of ∼0.44 g/g. The product yield increased to 0.53-0.62 g/g at a lower pH of 5.0-5.5, which, however, decreased the productivity to 1.28 g/L h. A higher final propionic acid titer of >55 g/L with a productivity of 2.23 g/L h was obtained in fed-batch HCD fermentation at pH 6.5. A 3-stage simulated fed-batch process in serum bottles produced 49.2 g/L propionic acid with a yield of 0.53 g/g and productivity of 0.66 g/L h. These productivities, yields and propionic acid titers were among the highest ever obtained in free-cell propionic acid fermentation.

Comportamento ingestivo de vacas leiteiras alimentadas com silagem de milheto ou milho / Ingestive behavior of dairy cattle fed millet and corn silage

Objetivou-se com este estudo avaliar o comportamento ingestivo de vacas leiteiras alimentadas com dietas contendo como volumoso silagem de milho ou silagem de milheto com 5 ou 20 mm de tamanho de partículas, com ou sem inoculante bacteriano. Foram utilizadas cinco vacas mestiças Holandês × Gir, com aproximadamente 100 dias de lactação ao início do experimento e peso corporal aproximado de 550 kg, produzindo, em média, 15,2 ± 2,3 kg de leite por dia. Os animais foram arranjados num delineamento em quadrado latino 5×5. O tempo total de alimentação, ruminação e ócio, não foi alterado pelo tipo de silagem na dieta. As variáveis que compõem o comportamento ingestivo não foram afetadas quando as vacas receberam as diferentes silagens de milheto comparadas com silagem de milho. O tamanho de partículas entre 5 e 20 mm, a presença ou não de inoculante e o tipo de silagem com teores de MS em 28%, não afeta o tempo total de alimentação, ruminação e ócio em vacas mestiças Holandês Gir com média de produção de 15,2 kg por dia. Silagem de milho ou milheto, tamanho de partículas 5 ou 20 mm, e o uso ou não de inoculante em silagem de milheto, não altera o tempo total despendido com alimentação, ruminação e ócio em dietas para vacas leiteiras mestiças Holandês × Gir com média de produção de 15,2 kg por dia.

The objective of this study was to evaluate the ingestive behavior of dairy cows fed corn and millet silage with a 5 mm particle size without inoculant, millet silage with a 5 mm particle size with inoculant, millet silage with a 20 mm particle size without inoculant, millet silage with a 20 mm particle size with inoculant. Five cows Holstein x Gir, with approximately 100 days of lactation at the beginning of the experiment and a mean body weight of 550 kg, producing an average of 15 kg of milk per day were used. The animals were arranged in a randomized 5 × 5 Latin square. The total feeding time, rumination time, chewing time, number of alimentary bolus, rumination time for bolus and number of chews was determined. The variables that make up the feeding behavior were not affected when the cows were fed different millet silages compared with corn silage. Cows fed corn silage and millet silage of 5 mm particle size had a higher intake of dry matter and neutral detergent fiber. A particle size between 5 and 20 mm, the presence or absence of inoculum and the type of silage with DM at 28%, does not affect the total feeding time , ruminating and resting of cows with an average production of 15.2 kg per day. The presence of the inoculant does not affect the intake of dry matter and neutral detergent fiber. Cows fed diets containing corn silage or pearl millet silage with a 5 mm particle size fed more DM.