Evaluation of industrial Saccharomyces cerevisiae strains as the chassis cell for second-generation bioethanol production

To develop a suitable Saccharomyces cerevisiae industrial strain as a chassis cell for ethanol production using lignocellulosic materials, 32 wild-type strains were evaluated for their glucose fermenting ability, their tolerance to the stresses they might encounter in lignocellulosic hydrolysate fermentation and their genetic background for pentose metabolism. The strain BSIF, isolated from tropical fruit in Thailand, was selected out of the distinctly different strains studied for its promising characteristics. The maximal specific growth rate of BSIF was as high as 0.65 h⁻¹ in yeast extract peptone dextrose medium, and the ethanol yield was 0.45 g g⁻¹ consumed glucose. Furthermore, compared with other strains, this strain exhibited superior tolerance to high temperature, hyperosmotic stress and oxidative stress; better growth performance in lignocellulosic hydrolysate; and better xylose utilization capacity when an initial xylose metabolic pathway was introduced. All of these results indicate that this strain is an excellent chassis strain for lignocellulosic ethanol production.     

Production of lactic acid using a new homofermentative Enterococcus faecalis isolate

Lactic acid is an intermediate-volume specialty chemical for a wide range of food and industrial applications such as pharmaceuticals, cosmetics and chemical syntheses. Although lactic acid production has been well documented, improved production parameters that lead to reduced production costs are always of interest in industrial developments. In this study, we describe the production of lactic acid at high concentration, yield and volumetric productivity utilizing a novel homofermentative, facultative anaerobe Enterococcus faecalis CBRD01. The highest concentration of 182 g lactic acid l⁻¹ was achieved after 38 h of fed-batch fermentation on glucose. The bacterial isolate utilized only 2–13% of carbon for its growth and energy metabolism, while 87–98% of carbon was converted to lactic acid at an overall volumetric productivity of 5 g l⁻¹ h⁻¹. At 13 h of fermentation, the volumetric productivity of lactate production reached 10.3 g l⁻¹ h⁻¹, which is the highest ever reported for microbial production of lactic acid. The lactic acid produced was of high purity as formation of other metabolites was less than 0.1%. The present investigation demonstrates a new opportunity for enhanced production of lactic acid with potential for reduced purification costs.     

Nitrogen loss by anaerobic oxidation of ammonium in rice rhizosphere

Anaerobic oxidation of ammonium (anammox) is recognized as an important process for nitrogen (N) cycling, yet its role in agricultural ecosystems, which are intensively fertilized, remains unclear. In this study, we investigated the presence, activity, functional gene abundance and role of anammox bacteria in rhizosphere and non-rhizosphere paddy soils using catalyzed reporter deposition–fluorescence in situ hybridization, isotope-tracing technique, quantitative PCR assay and 16S rRNA gene clone libraries. Results showed that rhizosphere anammox contributed to 31–41% N₂ production with activities of 0.33–0.64 nmol N₂ g⁻¹ soil h⁻¹, whereas the non-rhizosphere anammox bacteria contributed to only 2–3% N₂ production with lower activities of 0.08–0.26 nmol N₂ g⁻¹ soil h⁻¹. Higher anammox bacterial cells were observed (0.75–1.4 × 10⁷ copies g⁻¹ soil) in the rhizosphere, which were twofold higher compared with the non-rhizosphere soil (3.7–5.9 × 10⁶ copies g⁻¹ soil). Phylogenetic analysis of the anammox bacterial 16S rRNA genes indicated that two genera of ‘Candidatus Kuenenia'' and ‘Candidatus Brocadia'' and the family of Planctomycetaceae were identified. We suggest the rhizosphere provides a favorable niche for anammox bacteria, which are important to N cycling, but were previously largely overlooked.     

Control of the selectivity of butyric acid production and improvement of fermentation performance withClostridium tyrobutyricum

Summary The parameters that control fermentation performance of butyrate production have been studied with a selected strain ofClostridium tyrobutyricum. Fed-batch supply of glucose increased productivity for butyrate. The ratio of butyrate to total acids was strongly influenced by the growth rate of the bacteria, acetate being produced along with butyrate at higher growth rates. In glucose-limited, fed-batch cultures, initially produced acetate was re-utilized, resulting in exclusive production of butyrate. In cultures with non-limiting glucose feeding, the butyrate concentration reached 42.5 g·1^–1 with a selectivity of 0.90, a productivity of 0.82 g·^–1 per hour and a yield of 0.36 g·g^–1 The effects of the mode of supply of glucose on the production of butyrate and acetate are discussed in relation with the energy requirements for cell growth.     

Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD⁺. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g⁻¹) and the lowest xylitol yield (0.05 g g⁻¹) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Ethanol production by Escherichia coli from detoxified lignocellulosic teak wood hydrolysates with high concentration of phenolic compounds

Teak wood residues were subjected to thermochemical pretreatment, enzymatic saccharification, and detoxification to obtain syrups with a high concentration of fermentable sugars for ethanol production with the ethanologenic Escherichia coli strain MS04. Teak is a hardwood, and thus a robust deconstructive pretreatment was applied followed by enzymatic saccharification. The resulting syrup contained 60 g l^–1 glucose, 18 g l^–1 xylose, 6 g l^–1 acetate, less than 0.1 g l^–1 of total furans, and 12 g l^–1 of soluble phenolic compounds (SPCs). This concentration of SPC is toxic to E. coli, and thus two detoxification strategies were assayed: (1) treatment with Coriolopsis gallica laccase followed by addition of activated carbon and (2) overliming with Ca(OH)₂. These reduced the phenolic compounds by 40% and 76%, respectively. The detoxified syrups were centrifuged and fermented with E. coli MS04. Cultivation with the overlimed hydrolysate showed a 60% higher volumetric productivity (0.45 g_ETOH l^–1 hr^–1). The bioethanol/sugar yield was over 90% in both strategies.     

Detection of Melamine in Feed Using Liquid-Liquid Extraction Treatment Combined with Surface-Enhanced Raman Scattering Spectroscopy

A rapid, selective, and sensitive method to determine the melamine content in animal feeds was developed using surface-enhanced Raman scattering spectroscopy on aggregated 55 nm Au nanoparticles with liquid–liquid extraction sample preparation. Butyl alcohol was used as the initial extraction solvent, and liquid–liquid extraction was performed twice using HCl (pH 3–4) and 6∶1 (v/v) n-butyl alcohol/ethyl acetate. The intensity of the matrix-based peak at 731 cm⁻¹ was set at 100 as a basis for the feeds, and the peak at 707 cm⁻¹ was the characteristic peak of melamine used in the calculations. Sufficient linearity was obtained in the range 2–10 µg·g⁻¹ (R ² = 0.991). Limits of detection and quantification in the feeds were 0.5 and 2 µg·g⁻¹, respectively. The recovery rates were 82.5–90.2% with coefficients of variation below 4.02%. This new protocol could be easily developed for the routine monitoring of on-site feed quality and market surveillance.     

Construction and Analysis of High-Ethanol-Producing Fusants with Co-Fermentation Ability through Protoplast Fusion and Double Labeling Technology

Double labeling of resistance markers and report genes can be used to breed engineered Saccharomyces cerevisiae strains that can assimilate xylose and glucose as a mixed carbon source for ethanol fermentation and increased ethanol production. In this study Saccharomyces cerevisiae W5 and Candida shehatae 20335 were used as parent strains to conduct protoplast fusion and the resulting fusants were screened by double labeling. High performance liquid chromatography (HPLC) was used to assess the ethanol yield following the fermentation of xylose and glucose, as both single and mixed carbon sources, by the fusants. Interestingly, one fusant (ZLYRHZ7) was demonstrated to have an excellent fermentation performance, with an ethanol yield using the mixed carbon source of 0.424 g g⁻¹, which compares with 0.240 g g⁻¹ (W5) and 0.353 g g⁻¹ (20335) for the parent strains. This indicates an improvement in the ethanol yield of 43.4% and 16.7%, respectively.     

In glucose-limited continuous culture the minimum substrate concentration for growth,smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii,an environmentally abundant bacterium

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters μ_max and K_s were determined for growth with glucose. For both strains, μ_max was determined in batch culture after different precultivation conditions. In the case of C. heintzii, μ_max was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h⁻¹ C. heintzii grew immediately with a μ_max of 0.17±0.03 h⁻¹. After five transfers in batch culture, μ_max had increased only slightly to 0.18±0.03 h⁻¹. A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D=0.075 h⁻¹ into glucose-excess batch medium E. coli grew only after an acceleration phase of ∼3.5 h with a μ_max of 0.52 h⁻¹. After 120 generations and several transfers into fresh medium, μ_max had increased to 0.80±0.03 h⁻¹. For long-term adapted chemostat-cultivated cells, a K_s for glucose of 15 μg l⁻¹ for C. heintzii, and of 35 μg l⁻¹ for E. coli, respectively, was determined in ¹⁴C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h⁻¹. Using the determined pure culture parameter values for K_s and μ_max, it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s_min). The values estimated for s_min were dependent on growth rate; at D=0.05 h⁻¹, it was 12.6 and 0 μg l⁻¹ for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h⁻¹, s_min for E. coli had to be raised to 34.9 μg l⁻¹ whereas s_min for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K_s value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s_min.     

In vitro selection of blackberry (Rubus fruticosus ‘Tupy’) plants resistant to Botrytis cinerea using gamma ray-irradiated shoot tips

Blackberry is an economically important crop in Mexico, and its yield is substantially reduced by gray mold, a disease caused by Botrytis cinerea. One of the means to obtain B. cinerea-resistant plants is gamma irradiation. Shoot tips of in vitro-micropropagated blackberry plants (Rubus fruticosus ‘Tupy’) were irradiated with five doses of Cobalt-60 gamma radiation (0, 15, 30, 45, and 60 Gy) and cultured on Murashige and Skoog basal medium containing 1.0 mg l⁻¹ benzylaminopurine and 0.06 mg l⁻¹ indole-3-butyric acid (MSB medium). After 28 days of culture, survival was evaluated to determine mean lethal dose (LD₅₀), and 200 shoots were further irradiated at the determined LD₅₀ (30.8 Gy). After 28 days, the surviving shoots were micropropagated on MSB medium for 60 days. Non-irradiated shoots were screened for the in vitro selection of resistant B. cinerea, exposing them to different concentrations of sterile culture filtrate of B. cinerea (0, 2, 4, 6, 8, and 10 g l⁻¹) for 28 days to determine mean lethal concentration (LC₅₀), and the irradiated surviving shoots were further exposed to the determined LC₅₀ (4.6 g l⁻¹). Three surviving lines (rfgum5, rfgum6, and rfgum17) that did not present changes compared with the control shoots were micropropagated to obtain plantlets, which were further subjected to in vitro resistance assays using detached leaves inoculated with B. cinerea (1×10³ spores ml⁻¹). Plants of rfgum5 and rfgum6 mutant lines were highly resistant and presented similar growth to control plants. Therefore, this methodology is useful to obtain B. cinerea-resistant blackberry plants.     

Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h⁻¹ g (dry weight) of cells⁻¹ (0.24 to 0.30 g h⁻¹ g [dry weight] of cells⁻¹) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h⁻¹. The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h⁻¹ g (dry weight) of cells⁻¹ when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

Production,purification and biochemical characterisation of a novel lipase from a newly identified lipolytic bacterium Staphylococcus caprae NCU S6

A novel lipase, SCNL, was isolated from Staphylococcus caprae NCU S6 strain in the study. The lipase was purified to homogeneity with a yield of 6.13% and specific activity of 502.76 U/mg, and its molecular weight was determined to be approximately 87 kDa. SCNL maintained above 80% of its initial activity at a wide range of temperatures (20–50 °C) and pH values (6–11), with an optimal temperature at 40 °C and optimal pH at 9.0 with p-nitrophenyl palmitate as a substrate. SCNL exhibited a higher residual activity than the other staphylococcal lipases in the presence of common enzyme inhibitors and commercial detergents. The lipase activity was enhanced by organic solvents (isooctane, glycerol, DMSO and methanol) and metal ions (Na⁺, Ba²⁺, Ca²⁺, and Mn²⁺). The Km and Vmax values of SCNL were 0.695 mM and 262.66 s⁻¹mM⁻¹, respectively. The enzyme showed a preference for p-NP stearate, tributyrin and canola oil. These biochemical features of SCNL suggested that it may be an excellent novel lipase candidate for industrial and biotechnological applications.     

Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed‐batch and continuous cultures

Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the Calvin–Benson–Bassham (CBB) cycle into value-added chemical compounds by non-photosynthetic microorganisms. In this work, organoautotrophic growth of Ralstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization. The maximal growth yield was determined at 0.16 Cmole Cmole⁻¹ in a formate-limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH-controlled fed-batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed-batch cultures were also used to determine the maximum specific growth rate (μ_max = 0.18 h⁻¹) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l⁻¹.     

Biosynthesis of Poly-β-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava

The potential of Pseudomonas pseudoflava to produce poly-β-hydroxyalkanoates (PHAs) from pentoses was studied. This organism was able to use a hydrolysate from the hemicellulosic fraction of poplar wood as a carbon and energy source for its growth. However, in batch cultures, growth was inhibited completely at hydrolysate concentrations higher than 30% (vol/vol). When P. pseudoflava was grown on the major sugars present in hemicelluloses in batch cultures, poly-β-hydroxybutyric acid (PHB) accumulated when glucose, xylose, or arabinose was the sole carbon source, with the final PHB content varying from 17% (wt/wt) of the biomass dry weight on arabinose to 22% (wt/wt) of the biomass dry weight on glucose and xylose. Specific growth rates were 0.58 h⁻¹ on glucose, 0.13 h⁻¹ on xylose, and 0.10 h⁻¹ on arabinose, while the specific PHB production rates based on total biomass ranged from 0.02 g g⁻¹ h⁻¹ on arabinose to 0.11 g g⁻¹ h⁻¹ on glucose. PHB weight-average molecular weights were 640,000 on arabinose and 1,100,000 on glucose and xylose. The absolute amount of PHB in the cells decreased markedly when nitrogen limitation was relaxed by feeding ammonium sulfate at the end of the PHB accumulation stage of the arabinose and xylose fermentations. Copolymers of β-hydroxybutyric and β-hydroxyvaleric acids were produced when propionic acid was added to shake flasks containing 10 g of glucose liter⁻¹. The β-hydroxyvaleric acid monomer content attained a maximum of 45 mol% when the initial propionic acid concentration was 2 g liter⁻¹.     

Effect of controlled substrate feeding on butyric acid production byClostridium tyrobutyricum

Summary Production of butyric acid from wheat flour hydrolysate was studied withClostridium tyrobutyricum. The mode of substrate supply was found a key parameter for fermentation performance as large improvements were obtained by feeding with a non-limiting supply of substrate. With this procedure, increases in product concentration and productivity but also in selectivity and yield for butyrate were obtained. Substrate feeding controlled by the rate of gas production was found preferable to constant rate feeding for reason of convenience and flexibility. In these conditions, a butyrate concentration of 62.8 gl^–1 was obtained with a productivity of 1.25 gl^–1 h^–1, a selectivity of 91.5% and a yield of 0.45 g per g of glucose.     

Butyric acid fermentation in a fibrous bed bioreactor with immobilized Clostridium tyrobutyricum from cane molasses

Butyrate fermentation by immobilized Clostridium tyrobutyricum was successfully carried out in a fibrous bed bioreactor using cane molasses. Batch fermentations were conducted to investigate the influence of pH on the metabolism of the strain, and the results showed that the fermentation gave a highest butyrate production of 26.2 g l⁻¹ with yield of 0.47 g g⁻¹ and reactor productivity up to 4.13 g l⁻¹ h⁻¹ at pH 6.0. When repeated-batch fermentation was carried out, long-term operation with high butyrate yield, volumetric productivity was achieved. Several cane molasses pretreatment techniques were investigated, and it was found that sulfuric acid treatment gave better results regarding butyrate concentration (34.6 ± 0.8 g l⁻¹), yield (0.58 ± 0.01 g g⁻¹), and sugar utilization (90.8 ± 0.9%). Also, fed-batch fermentation from cane molasses pretreated with sulfuric acid was performed to further increase the concentration of butyrate up to 55.2 g l⁻¹.     

Efficient Open Fermentative Production of Polymer-Grade L-Lactate from Sugarcane Bagasse Hydrolysate by Thermotolerant Bacillus sp. Strain P38

Lactic acid is one of the top 30 potential building-block chemicals from biomass, of which the most extensive use is in the polymerization of lactic acid to poly-lactic-acid (PLA). To reduce the cost of PLA, the search for cheap raw materials and low-cost process for lactic acid production is highly desired. In this study, the final titer of produced L-lactic acid reached a concentration of 185 g·L⁻¹ with a volumetric productivity of 1.93 g·L⁻¹·h⁻¹ by using sugarcane bagasse hydrolysate as the sole carbon source simultaneously with cottonseed meal as cheap nitrogen sources under the open fed-batch fermentation process. Furthermore, a lactic acid yield of 0.99 g per g of total reducing sugars was obtained, which is very close to the theoretical value (1.0 g g⁻¹). No D-isomer of lactic acid was detected in the broth, and thereafter resulted in an optical purity of 100%, which exceeds the requirement of lactate polymerization process. To our knowledge, this is the best performance of fermentation on polymer-grade L-lactic acid production totally using lignocellulosic sources. The high levels of optically pure l-lactic acid produced, combined with the ease of handling and low costs associated with the open fermentation strategy, indicated the thermotolerant Bacillus sp. P38 could be an excellent candidate strain with great industrial potential for polymer-grade L-lactic acid production from various cellulosic biomasses.     

Human milk oligosaccharides shorten rotavirus-induced diarrhea and modulate piglet mucosal immunity and colonic microbiota

The impact of human milk oligosaccharides (HMO) on mucosal immunity, gut microbiota and response to rotavirus (RV) infection was investigated in the piglet model. Newborn piglets were fed with formula alone (FF) or formula supplemented with 4 g l⁻¹ HMO (HMO) or a prebiotic mixture of 9:1 short-chain galactooligosaccharides (3.6 g l⁻¹) and long-chain fructooligosaccharides (0.4 g l⁻¹) (PRE) (n=19–21 per group) for 15 days. Piglets (n=7–8) in each dietary group were orally infected with porcine rotavirus (RV) OSU strain on d10, and stool consistency was assessed daily. Blood, small intestine and colonic contents were collected at day 15. Serum RV-specific antibody concentrations, intestinal histomorphology, RV non-structural protein-4 (NSP4) and cytokine mRNA expression were assessed. Colonic content pH, dry matter (DM) and short-chain fatty acid concentrations were measured. Ascending colonic microbiota was analyzed by 16S rRNA gene v1-3 region pyrosequencing. HMO- and PRE-fed groups had shorter duration of diarrhea than FF piglets. Infection changed intestinal histomorphology, increased serum RV-specific antibody response and intestinal RV NSP4 expression, and modulated ileal cytokine expression. HMO enhanced T helper type 1 (interferon-gamma) and anti-inflammatory (interleukin-10) cytokines in the ileum, while prebiotics promoted RV-specific immunoglobulin M response to the infection. RV infection and HMO supplementation altered intraluminal environment and gut microbiota. HMO increased pH and lowered DM of colonic contents and enhanced the abundance of unclassified Lachnospiraceae, which contains numerous butyrate-producing bacteria. In conclusion, HMO and prebiotics did not prevent the onset of RV infection but reduced the duration of RV-induced diarrhea in piglets, in part, by modulating colonic microbiota and immune response to RV infection.