Fermentation End Products Research Articles

Abstract 2831: Detection of methane-producing archaea in the cervix of Hispanics using shotgun metagenomics

Abstract The human cervix is colonized by microbial communities that have traditionally been regarded as a protective barrier against infections. Dysbioses disrupting the balance of the normal microbiota, leads to the appearance of anaerobic taxa that have been related to sexual transmitted infections and cancer. The complexity of the diversity and composition of cervicovaginal communities has been recently highlighted by 16S rDNA surveys, although these have limitations to either select mostly bacteria and the lack of strong lower taxonomic resolution such as being species-specific. Metagenomic shotgun sequencing overcomes these limitations, and gives us access to the extent of diversity beyond bacterial communities. This is the first project to characterize the bacterial and archaeal communities of the cervix of Puerto Ricans using shotgun metagenomics. Women coming for gynecology and colposcopy evaluation at the UPR and San Juan City clinics (San Juan Metropolitan area), and who did not meet several excluding criteria including having taken antibiotics for the last month, were recruited for the study. Samples were acquired by inserting a speculum for access and visualization of the cervix and a sterile swab was rotated along the lumen with a circular motion and frozen immediately for posterior genomic DNA extractions and sequencing. Library construction and shotgun sequencing of 10 cervical patient samples was done with the Illumina HiSeq platform An average of 1,690,000 good quality reads per sample were analyzed. Approximately 90% of the contigs had more than 1,000bp and 10% had sizes ranging from 1,000-5,000bp. Microbial communities were dominated by Lactobacillus, including L. iners, Gardnerella vaginallis, Nocardia and Atapobium vaginae. Archaeal diversity was dominated by Methanosarcina (namely, M. barkeri) with other less dominant taxa such as Methanolobus, Methanococcus, Natrococcus and Methanobrevibacter. The dominance of Lactobacillus was expected, and as Archaea are environmental organisms that have been associated with the mucosa in mammals, including protecting the human gut, they must have an important role in the cervix. As methanogenic archaea are known to remove fermentation end products, such as methanol and ethanol, as end-products of other fermentative bacteria, methanogenesis maybe important in preventing the accumulation of gases and other reaction end products that could damage the cervical mucosa. Citation Format: Filipa Godoy-Vitorino, Frances Vazquez-Sanchez, Anelisse Dominicci-Maura, Josefina Romaguera. Detection of methane-producing archaea in the cervix of Hispanics using shotgun metagenomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2831.

Recent advances in the feeding and nutrition of dairy goats.

There have been recent advances concerning research of the feeding and nutrition of dairy goats in a wide array of areas. Ruminally emitted methane and supplementary feedstuffs to a lesser extent make appreciable contributions to the carbon footprint of dairy goats, with the former affected by type of production system and associated dietary characteristics. Unique behavior of goats necessitates careful consideration of the nature of confinement facilities to achieve optimal production by animals differing in social hierarchy. Physiological conditions such as nutritional needs and perhaps health status may influence diet selection by goats in both grazing and confinement settings. Some research suggests that low concentrations of protein and fat in milk of high-yielding dairy goat breeds could involve the type and nature of dietary ingredients as influencing end products of ruminal fermentation. With the relationship between milk urea nitrogen concentration and efficiency of dietary protein utilization, through future research the measure may be a useful tool for diet formulation as in dairy cattle. Effects of dietary inclusion of sources of fats and oils vary considerably depending on their nature, as is also true for byproduct feedstuffs and conventional ones being substituted for. Supplementation of dairy goats with sources of polyunsaturated fatty acids can affect oxidative stress and various feedstuffs influence antioxidant status; however, research addressing the significance of such changes under practical production settings would be beneficial.

Development of an analytical method to detect short-chain fatty acids by SPME-GC–MS in samples coming from an in vitro gastrointestinal model

Short chain fatty acids (SCFA) are end-products of intestinal bacterial fermentation. The concentrations of fermentation metabolites are closely related to the microbial activity that occurs in various digestive compartments. The fermentation products may vary qualitatively and quantitatively, especially within the colon. The Simulator of the Human Intestinal Microbial Ecosystem (SHIME), an in vitro dynamic and multicompartment model of the human intestinal tract, can be adapted to mimic the piglet gastrointestinal tract. In this context, a quantitative method, based on solid phase microextraction gas chromatography coupled to mass spectrometry (SPME–GC–MS), was developed for the determination of seven short chain fatty acids, i.e. acetic, propionic, butyric, isobutyric, valeric, isovaleric and hexanoic acids, in samples coming from this experimental in vitro gastrointestinal model. The advantage of the SPME-GC–MS technique is that the seven compounds could be determined in a single run, after a simple and rapid sample treatment, without any other extraction than the automatic SPME. The developed method was validated in accordance to the European and US FDA guidelines and showed good specificity/selectivity. In addition, limits of detection and quantification ranged from 8 to 72 mg L−1 and from 16 to 144 mg L−1, respectively. Two internal quality control samples spiked at different concentrations were analyzed to assess the trueness of the developed method, which ranged between 97.7 and 122.4% of the expected value, for the seven compounds analyzed. The method was successfully applied to twenty samples coming from a gastrointestinal model, with different inocula. The developed method might be used as a general method for measuring SCFA in biological samples.

Clostridium fungisolvens sp. nov., a new β-1,3-glucan-decomposing bacterium isolated from anoxic soil subjected to biological soil disinfestation.

Biological soil disinfestation (BSD) or reductive soil disinfestation (RSD) is a bioremediation method used to suppress or eliminate soil-borne plant pathogens by stimulating activities of indigenous anaerobic bacteria of the soil. An anaerobic bacterial strain (TW1T) was isolated from an anoxic soil sample subjected to the BSD treatment and comprehensively characterized. Cells of the strain were Gram-stain-positive, slightly curved and motile rods producing terminal spores. The strain was aerotolerant. Strain TW1T was saccharolytic and produced acetate, butyrate, H2 and CO2 as fermentation end products. Strain TW1T decomposed β-1,3-glucan (curdlan and laminarin) and degraded mycelial cells of an ascomycete Fusarium plant pathogen. Major cellular fatty acids of strain TW1T were C14 : 0, C14 : 0 dimethylacetal (DMA), C16 : 0 aldehyde and C16 : 0 DMA. Strain TW1T made a group on the phylogenetic tree constructed based on 16S rRNA gene sequences with species such as Clostridium fallax (96.3 %) and Clostridium polyendosporum (96.0 %). Whole genome analysis of strain TW1T showed that the total length of the genome was 5.28 Mb with the DNA G+C content of 31.3 mol%. The average nucleotide identity (ANIb) between strain TW1T and C. fallax was 71.2 %. Presence of the genes encoding laminarinase or GH16 β-glucosidase was confirmed from the genome analysis of strain TW1T. Based on the genomic, phylogenetic and phenotypic properties obtained, we propose strain TW1T should be assigned in the genus Clostridium in the family Clostridiaceae as Clostridium fungisolvens sp. nov. The type strain TW1T (=NBRC 112097T=DSM 110791T).

Clostridium vitabionis sp. nov., isolated from the large intestine of a mini-pig.

An obligately anaerobic, Gram-stain-negative, spore-forming, short rod-shaped bacterium, designated strain YH- T4B42T, was isolated from the large intestine of a mini-pig. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Clostridium and is most closely related to Clostridium aminophilum KCTC 5424T, Clostridium symbiosum KCTC 15329T and Clostridium butyricum KCTC 1871T, with 95.5, 92.4 and 83.0 % sequence similarity, respectively. The average nucleotide identity values for strain YH-T4B42T and the closest related strains were lower than 72 %. The G+C content of the isolate was 55.8 mol%. The cell-wall peptidoglycan was A1γ type and contained meso-diaminopimelic acid. The predominant fatty acids were C16 : 0, C18 : 1 cis 9, C14 : 0 and C18 : 0. The major end products of glucose fermentation were lactate, formate and acetate, with a minor amount of butyrate. Based on its phenotypic, phylogenetic and chemotaxonomic properties, a novel species, Clostridium vitabionis sp. nov., is proposed for strain YH-T4B42T (=KCTC 25105T=NBRC 114767T).

Sporofaciens musculi gen. nov., sp. nov., a novel bacterium isolated from the caecum of an obese mouse.

A bacterial strain, designated WCA-9-b2T, was isolated from the caecal content of an 18-week-old obese C57BL/6NTac male mouse. According to phenotypic analyses, the isolate was rod-shaped, strictly anaerobic, spore-forming, non-motile and Gram-stain-positive, under the conditions tested. Colonies were irregular and non-pigmented. Analysis of the 16S rRNA gene sequence indicated that the isolate belonged to the order Clostridiales with Dorea longicatena ATCC 27755T (94.9 % sequence identity), Ruminococcus gnavus ATCC 29149T (94.8%) and Clostridium scindens ATCC 35704T (94.3%) being the closest relatives. Whole genome sequencing showed an average nucleotide identity <74.23 %, average amino acid identity <64.52-74.67 % and percentage of conserved proteins values <50 % against the nine closest relatives (D. longicatena, Ruminococcus gnavus, C. scindens, Dorea formicigenerans, Ruminococcus lactaris, Clostridium hylemonae, Merdimonas faecis, Faecalicatena contorta and Faecalicatena fissicatena). The genome-based G+C content of genomic DNA was 44.4 mol%. The major cellular fatty acids were C16 : 0 (24.5%), C18 : 1 cis9 (19.8 %), C16 : 0 DMA (11.7%), C18 : 0 (8.4%) and C14 : 0 (6.6%). Respiratory quinones were not detected. The predominant metabolic end products of glucose fermentation were acetate and succinate. Production of CO2 and H2 were detected. Based on these data, we propose that strain WCA-9-b2T represents a novel species within a novel genus, for which the name Sporofaciens musculi gen. nov., sp. nov. is proposed. The type strain is WCA-9-b2T (=DSM 106039T=CECT 30156T).

Unveiling the relationships between diet composition and fermentation parameters response in dual-flow continuous culture system: a meta-analytical approach.

The objective of this study was to investigate the functional form of the relationship between diet composition (dietary crude protein [CP] and neutral detergent fiber [NDF]) and amount of substrate (fermenter dry matter intake [DMI]) with microbial fermentation end products in a dual-flow continuous culture system. A meta-analysis was performed using data from 75 studies. To derive the linear models, the MIXED procedure was used, and for nonlinear models, the NLMIXED procedure was used. Significance levels to fit the model assumed for fixed and random effects were P ≤ 0.05. Independent variables were dietary NDF, CP, and fermenter DMI, whereas dependent variables were total volatile fatty acids (VFA) concentration; molar proportions of acetate, propionate, and butyrate; true ruminal digestibilities of organic matter (OM), CP, and NDF; ammonia nitrogen (NH3–N) concentration and flows of NH3–N; non-ammonia nitrogen; bacterial-N; dietary-N; and efficiency of microbial protein synthesis (EMPS). Ruminal digestibilities of OM, NDF, and CP decreased as fermenter DMI increased (P < 0.04). Dietary NDF and CP digestibilities were quadratically associated (P < 0.01). Total VFA linearly increased as DMI increased (P < 0.01), exponentially decreased as dietary NDF increased (P < 0.01), and was quadratically associated with dietary CP (P < 0.01), in which total VFA concentration was maximized at 18% dietary CP. Molar proportion of acetate exponentially increased (P < 0.01) as dietary NDF increased. Molar proportion of propionate linearly increased and exponentially decreased as DMI and dietary NDF increased, respectively (P < 0.01). Bacterial-N quadratically increased and dietary-N exponentially increased as DMI increased (P < 0.01). Flows of bacterial-N and dietary-N linearly decreased as dietary NDF increased (P < 0.02), and dietary-N flow was maximized at 18% CP. The EMPS linearly increased as dietary CP increased (P < 0.02) and was not affected by DMI or dietary NDF (P > 0.05). In summary, increasing fermenter DMI increased total VFA concentration and molar proportion of propionate, whereas, dietary NDF increased the molar proportion of acetate. Dietary CP increased bacterial-N flow and was positively associated with NH3–N concentration. Overall, the analysis of this dataset demonstrates evidences that the dual-flow continuous culture system provides valuable estimates of ruminal digestibility, VFA concentration, and nitrogen metabolism.

Fecal short-chain fatty acid concentrations and dysbiosis in dogs with chronic enteropathy.

BackgroundAccumulating evidence shows an important relationship between the gastrointestinal (GI) microbiota and host health. Microbial metabolites are believed to play a critical role in host‐microbial interactions. Short‐chain fatty acids (SCFAs) are major end products of bacterial carbohydrate fermentation in the intestinal tract. Decreased concentrations of SCFAs have been observed in humans with GI disease. However, large‐scale clinical data in dogs are lacking.Hypothesis/ObjectiveTo evaluate fecal concentrations of SCFAs and the fecal microbiota in healthy control (HC) dogs and dogs with chronic enteropathy (CE).AnimalsForty‐nine privately owned HC dogs and 73 dogs with CE.MethodsProspective cohort study. Fecal concentrations of SCFAs were measured using gas chromatography/mass spectrometry. Illumina sequencing and quantitative real‐time polymerase chain reaction were utilized to evaluate the fecal microbiota.ResultsFecal concentrations (median [range] μmol/g of dry matter) of acetate were lower (P = .03) in dogs with CE (185.8 [20.1‐1042.1]) than in HC dogs (224.0 [87.7‐672.8]). Propionate were also lower (P < .001) in dogs with CE (46.4 [0.4‐227.9]) than in HC dogs (105.9 [1.6‐266.9]). Moreover, total SCFAs were lower (P = .005) in dogs with CE (268.1 [21.8‐1378.2]) than in HC dogs (377.2 [126.6‐927.0]). Dysbiosis in dogs with CE was characterized by decreased bacterial diversity and richness, distinct microbial community clustering compared with that in HC dogs, and a higher dysbiosis index.Conclusions and Clinical ImportanceDogs with CE had an altered fecal SCFA concentration accompanied by significant changes of the fecal microbiota.

Methane Production of Fresh Sainfoin, with or without PEG, and Fresh Alfalfa at Different Stages of Maturity is Similar but the Fermentation End Products Vary.

Simple SummaryIn the last years, there has been increasing interest in the use of forages containing condensed tannins (CT) in ruminant nutrition. Condensed tannins can reduce the methane emissions and the ruminal degradation of protein, improving the animal performances to different extents depending on the source and dose of CT. In vitro fermentation of sainfoin has not been studied in fresh forage. The effect of CT can be studied in comparison with a similar CT-free forage or using polyethylene glycol (PEG), which is a tannin-blocking agent. The maturity stage influences the chemical composition to a different degree depending on the legume species, and can affect the content and fractions of CT. The aims of this trial were to compare the fermentation parameters of sainfoin with or without PEG, to detect the differences due to CT, at different maturity stages (vegetative, start-flowering, and end-flowering) and compare them with the fermentation parameters of alfalfa. The main results were that sainfoin had greater in vitro organic matter degradability (IVOMD) and lower ammonia and acetic:propionic ratio than alfalfa. Sainfoin CT had effect on ammonia and individual fatty acid proportions. In conclusion, fermentation end products were affected both by the chemical composition and CT contents. Alfalfa and sainfoin are high-quality forages with different condensed tannins (CT) content, which can be affected by the stage of maturity. To study the effects of CT on fermentation parameters, three substrates (alfalfa, sainfoin, and sainfoin+PEG) at three stages of maturity were in vitro incubated for 72 h. Sainfoin had greater total polyphenol and CT contents than alfalfa. As maturity advanced, CT contents in sainfoin decreased (p < 0.05), except for the protein-bound CT fraction (p > 0.05). The total gas and methane production was affected neither by the substrate nor by the stage of maturity (p > 0.05). Overall, sainfoin and sainfoin+PEG had greater in vitro organic matter degradability (IVOMD) than alfalfa (p < 0.05). Alfalfa and sainfoin+PEG presented higher ammonia content than sainfoin (p < 0.001). Total volatile fatty acid (VFA) production was only affected by the stage of maturity (p < 0.05), and the individual VFA proportions were affected by the substrate and the stage of maturity (p < 0.001). In conclusion, alfalfa and sainfoin only differed in the IVOMD and the fermentation end products. Moreover, CT reduced ammonia production and the ratio methane: VFA, but the IVOMD was reduced only in the vegetative stage.

Shifts in fermentation end products and bacterial community composition in long-term, sequentially transferred in vitro ruminal enrichment cultures fed switchgrass with and without ethanol as a co-substrate

In vitro ruminal fermentations resemble in vivo fermentations with respect to substrate consumption and distribution of fermentation products in short term (1–5 d) incubations. However, little is known regarding changes in in vitro fermentations over prolonged incubation or multiple transfers. Gas production, pH, fermentation products, and bacterial community composition were examined in duplicate in vitro fermentations of switchgrass plus distillers grains that were transferred at 3–4 d intervals over 900 d. Additionally, duplicate fermentations inoculated from 160 d-old enrichments into the same medium but supplemented with ethanol, and transferred at 3–4 d over a 730 d period were characterized. SWG and SWG + E fermentation showed marked differences in community composition, pH, total product concentrations and ratios, relative to each other and to the original inoculum. The results have implications for the use of ruminal inocula for industrial production of short- and medium-chain fatty acids via the carboxylate platform.

Personalizing Food to the Gut Microbiome

Dietary polysaccharides from plants nourish human gut bacterial communities (microbiota). These carbohydrates promote the growth of resident microbes that resist pathogens, metabolize drugs, and train the immune system. Short‐chain fatty acids, which are end products of microbial polysaccharide fermentation, are also crucial metabolic precursors and energy sources for human colon cells. Yet, despite growing awareness for the importance of polysaccharides on gut bacterial growth and metabolism, optimal strategies for consuming these carbohydrates have lagged. Multiple sources of dietary carbohydrates (prebiotics) exist and each has varying effects on gut bacterial species. Moreover, gut microbial communities vary widely between people and even within individuals over time. Here, we describe our efforts to understand how prebiotic treatments should account for inter‐individual differences in gut microbiota composition. These efforts rely on experimental tools including high‐throughput bacterial culture assays and artificial gut systems. Using these systems, we find evidence for prebiotic‐ and individual‐specific components of gut microbiota responses to prebiotic therapy. We also observe changing prebiotic responses within individual gut microbiota over time. Lastly, our experiments suggest the potential for developing diagnostics to predict individual prebiotic response.Support or Funding InformationLAD acknowledges support from the Global Probiotics Council, a Searle Scholars Award, the Hartwell Foundation, an Alfred P. Sloan Research Fellowship, the Translational Research Institute through Cooperative Agreement NNX16AO69A, the Damon Runyon Cancer Research Foundation, the Hartwell Foundation, and the NIH (1R01DK116187‐01, P30 DK034987).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Supplementation of Piper sarmentosum Leaf Powder on Feed Efficiency, Rumen Ecology and Rumen Protozoal Concentration in Thai Native Beef Cattle.

Simple SummaryThis work was to study the influence of Piper sarmentosum (PS) leaf powder supplementation in cattle diet. It was found that supplementation of PS leaf powder at 2.4 g/d resulted in improving total feed intake and dry matter (DM) digestibility whereas there was reduced protozoal population and methane (CH4) production in cattle.sThe aim of this research was to study the influence of inclusion of Piper sarmentosum (PS) leaf powder on feed efficiency, rumen ecology, protozoal diversity and CH4 emission in cattle. Four male beef cattle (1–1.5 years old) with similar initial body weights (BW) of 150 ± 20.00 kg were randomly assigned to a 4 × 4 Latin squared design. Experimental diets consisted of four levels of PS leaf powder supplementation at 0 (control), 0.6, 1.2 and 2.4 g/head/d, respectively. Dry matter (DM) intake of rice straw and total intake in terms of g/kg BW0.75 differed among treatments and were linearly greatest when PS was added at 2.4 g/head/d (p < 0.05). PS leaf powder did not affect the intake of nutrients and digestibility of organic matter, crude protein, and fibers. Nevertheless, digestibility of DM was found to increase by 4.8% in cattle when added at 2.4 g/head/d as compared to the other control treatments. PS leaf powder did not affect ruminal pH, ruminal temperature, ammonia–nitrogen concentration and blood urea-nitrogen (p > 0.05). The bacterial population was similar across PS levels (p > 0.05). However, the protozoal count was lower in animals fed with the supplement at 2.4 g/head/d (p < 0.05). The PS leaf powder did not affect total volatile fatty acid (VFA) and acetic acid (C2), butyric acid (C4), C2 to propionic acid (C3) and C2 to C3 to C4. However, concentration of C3 at 4 h post feeding and mean value linearly increased with supplementation of PS leaf powder at 2.4 g/d. The 4 h post-feeding and mean values of CH4 concentration were linearly reduced with PS supplementation (p < 0.05). An increase of PS leaf powder at 2.4 g decreased CH4 after feeding 4 h by 21.33% when compared to no PS leaf powder. Thus, supplementation of PS leaf powder at 2.4 g in ruminant feeding is recommended for manipulating rumen efficiency.

Evaluation and Optimization of Sample Handling Methods for Quantification of Short-Chain Fatty Acids in Human Fecal Samples by GC-MS.

The gut microbiota has attracted a great deal of interest in recent years due to its association with many diseases. Short-chain fatty acids (SCFAs), the end products of dietary fiber fermentation by the intestinal microbiota, are among the most frequently discussed gut metabolites. As the sample handling method greatly affects the integrity of data, this study investigated the most important parameters that affect the bias of SCFA comparisons in human fecal studies. An accurate gas chromatography-mass spectrometry (GC-MS) method was first established and validated for quantifying six SCFAs, including acetic, propionic, butyric, isobutyric, isovaleric, and valeric acids. To remove interfering species, we used butanol to extract SCFAs from acidified fecal suspensions. The validated quantification method was then applied to evaluate fecal sample handling protocols. We found that lyophilization of fecal samples can not only minimize bias due to the water content but also provide better stability of SCFAs. Six SCFAs were stable and that their recoveries were higher than 90% after lyophilization. Lyophilization of a large fecal sample is extremely time-consuming, and 1 g of fecal sample is suggested for lyophilization to minimize sampling bias. The interindividual difference was significantly higher than the intra-individual difference when using 1 g of fecal sample to study SCFAs. Finally, an effective protocol from sample collection to GC-MS analysis was proposed. As SCFAs have been shown to play an important role in health maintenance and disease development, the proposed protocol is anticipated to be applicable to clinical studies to delineate the biological functions of each SCFA.

Comparison of nitrogen transformation dynamics in non-irradiated and irradiated alfalfa and red clover during ensiling.

ObjectiveTo study the contribution of plant enzyme and microbial activities on protein degradation in silage, this study evaluated the nitrogen transformation dynamics during ensiling of non- and irradiated alfalfa (Medicago sativa L.) and red clover (Trifolium pratense L.).MethodsAlfalfa and red clover silages were prepared and equally divided into two groups. One group was exposed to γ-irradiation at a recommended dosage (25 Gky). Therefore, four types of silages were produced: i) non-irradiated alfalfa silage; ii) irradiated alfalfa silage; iii) non-irradiated red clover silage; and iv) irradiated red clover silage. These silages were opened for fermentation quality and nitrogen components analyses after 1, 4, 8, and 30 days, respectively.ResultsThe γ-irradiation successfully suppressed microbial activity, indicated by high pH and no apparent increases in fermentation end products in irradiated silages. All nitrogen components, except for peptide-N, increased throughout the ensiling process. Proteolysis less occurred in red clover silages compared with alfalfa silages, indicated by smaller (p<0.05) increment in peptide-N and free amino acid N (FAA-N) during early stage of ensiling. The γ-irradiation treatment increased (p<0.05) peptide-N and FAA-N in alfalfa silage at day 1, whereas not in red clover silage; these two nitrogen components were higher (p<0.05) between day 4 and day 30 in non-irradiated silages than the irradiated silages. The ammonia nitrogen and non-protein nitrogen were highest in non-irradiated alfalfa silage and lowest in irradiated red clover silage after ensiling.ConclusionThe result of this study indicate that red clover and alfalfa are two forages varying in their nitrogen transformation patterns, especially during early stages of ensiling. Microbial activity plays a certain role in the proteolysis and seems little affected by the presence of polyphenol oxidase in red clover compared with alfalfaa.

Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis

Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays.Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.

Determination of Glucose Concentration in Anaerobic Acidification Cultures by Portable Glucose Monitoring System

In mixed microbial fermentation, sugar concentration should be monitored regularly in order to evaluate the effectiveness of fermentation process. Anaerobic acidification fermentation is the process involving microbes to convert the soluble carbohydrate (e.g. glucose) derived from the hydrolysis of insoluble carbohydrates (e.g. starch). The determination of glucose during the fermentation is essential in order to evaluate the mass balance of electron transfer from the oxidation of glucose to the fermentation end-products (e.g. organic acids and alcohols). The fast and practical measurements of glucose concentration in the fermentation broth are highly required to evaluate and ensure the stability of fermentation process. The results showed that once glucose as a soluble sugar is available in the fermentation broth, it was accurately measured by GlucoDr blood glucose biosensor. The standard curve of glucose solution showed the linear relationship between glucometer reading and glucose concentration in which the coefficient determination obtained was at about 0.99. This indicated that glucose analysis with using GlucoDr blood glucose biosensor was accurate and reproducible.

Acetone-butanol-ethanol solvents improved enzymatic hydrolysis of pretreated energy grass

Simultaneous saccharification and fermentation (SSF) is a promising process for conversion of lignocellulosic materials to biofuels. However, the fermentation end products might inhibit the saccharification of lignocellulosics by cellulose-degrading enzymes. This study investigated the effects of acetone-butanol-ethanol (ABE) fermentation products on enzymatic hydrolysis of energy grasses. After addition of 5 g/L butanol during the hydrolysis, the glucose yields of pretreated Hybrid Pennisetum and Avicel apparently increased from 62.8% and 33.4% to 77.5% and 63.8%, respectively. The acetone and ethanol also showed the promotional effect at higher concentrations (>10 g/L), but the acetic acid and butyric acid exhibited slight inhibition on the hydrolysis. The accumulative effect of these solvents was observed by addition of solvent mixtures. The ABE solvents improved the activity of cellobiohydrolase (CBH) I and endoglucanase (EG) II remaining in hydrolysis solution, particularly butanol increased the activity of EG II to 142.8%, which probably contributed to the enhanced hydrolysis. Thus, the inhibition of ABE end products on hydrolysis of lignocellulosic materials during SSF was negligible, but instead, the promotion on enzymatic hydrolysis provided an opportunity for effective utilization of enzymes.

Intestinibaculum porci gen. nov., sp. nov., a new member of the family Erysipelotrichaceae isolated from the small intestine of a swine.

A strictly anaerobic, Gram-stain-positive, catalase-negative, non-motile, rod-shaped bacterium, designated SG0102T, was isolated from the small intestine of a swine. Optimal growth occurred at 37°C and pH 7.0. Furthermore, growth was observed in the presence of up to 3% (w/v) NaCl but not at salinity levels higher than 4%. The comparative analysis of 16S rRNA gene sequences showed that strain SG0102T was most closely related to Kandleria vitulina DSM 20405T (93.3%), followed by Catenibacterium mitsuokai KCTC 5053T (91.1%), Sharpea azabuensis KCTC 15217T (91.0%), and Eggerthia catenaformis DSM 5348T (89.6%). The average nucleotide identity values between strain SG0102T and related species, K. vitulina DSM 20405T, C. mitsuokai KCTC 5053T, S. azabuensis KCTC 15217T, and E. catenaformis DSM 5348T, were 71.0, 69.3, 70.0, and 69.2%, respectively. The phylogenetic analysis based on 16S rRNA gene sequence revealed that strain SG0102T belonged to the family Erysipelotrichaceae in the class Erysipelotrichia. The DNA G + C content of the strain SG0102T was 39.5 mol%. The major cellular fatty acids (> 10%) of strain SG0102T were C16:0, C16:0 dimethyl acetal, and C18:2ω9/12c. The cell wall peptidoglycan of strain SG0102T contained the meso-diaminopimelic acid. The strain SG0102T produced lactic acid as a major end product of fermentation. These distinct phenotypic and phylogenetic properties suggest that strain SG0102T represents a novel species in a novel genus of the family Erysipelotrichaceae, for which the name Intestinibaculum porci gen. nov. sp. nov. is proposed. The type strain is SG0102T (= KCTC 15725T = NBRC 113396T).

Effects of a Saccharomyces cerevisiae fermentation product on fecal characteristics, nutrient digestibility, fecal fermentative end-products, fecal microbial populations, immune function, and diet palatability in adult dogs1.

Yeast products may serve as functional ingredients due to their benefits on host health but vary greatly in source, composition, and functionality, justifying research in host species of interest. In this study, a Saccharomyces cerevisiae fermentation product (SCFP) was investigated as a dietary supplement for adult dogs. Adult female beagles (n = 12; mean age = 3.3 ± 0.8 yr; mean BW = 10.3 ± 0.68 kg) were fed the same diet, but supplemented with three levels of SCFP (125, 250, and 500 mg/d) or a placebo (sucrose) via gelatin capsules in a replicated 4 × 4 Latin square design. Fecal samples for nutrient digestibility, fecal characteristics and microbial populations as well as blood samples for immune indices were collected after a 21-d adaptation phase in each period. A separate palatability test was conducted to examine palatability of an SCFP-containing diet (0.2% of diet). All data, except for palatability data, were analyzed by Mixed Models procedure of SAS (version 9.4). A paired t-test was conducted to analyze data from the palatability test. Supplementation of SCFP did not affect total tract apparent macronutrient and energy digestibilities or fecal characteristics. Fecal phenol and total phenol + indole concentrations decreased linearly with SCFP dosage (P < 0.05). Relative abundance of Bifidobacterium was greater (P < 0.05), while Fusobacterium was lower (P < 0.05) in SCFP-supplemented dogs. Total white blood cell counts were decreased by SCFP (P < 0.05). The percentage of natural killer cells and antigen-presenting cells were not altered by SCFP. However, when comparing control vs. all SCFP treatments, SCFP-supplemented dogs had greater (P < 0.05) major histocompatibility complex class II presenting B cell and monocyte populations than control dogs. IFN-γ secreting helper and cytotoxic T cells increased linearly with SCFP consumption (P < 0.05). Immune cells derived from SCFP-supplemented dogs produced less (P < 0.05) TNF-α than those from control dogs when cells were stimulated with agonists of toll-like receptors 2, 3, 4, and 7/8. A linear increase (P < 0.05) in serum IgE with SCFP dosage was noted. In the palatability test, a 1.9:1 consumption ratio was observed for the SCFP-containing diet vs. control diet, demonstrating a preference (P < 0.05) for SCFP. Results of this study suggest that SCFP supplementation may be beneficial to adult dogs by positively altering gut microbiota, enhancing immune capacity and reducing inflammation.

Sex Differences in Pulmonary Responses to Ozone in Mice. Role of the Microbiome.

We have previously reported that the mouse gut microbiome contributes to pulmonary responses to ozone, a common asthma trigger, and that short-chain fatty acids, end products of bacterial fermentation, likely contribute to this role of the microbiome. A growing body of evidence indicates that there are sex-related differences in gut microbiota and these differences can have important functional consequences. The purpose of this study was to determine whether there are sex-related differences in the impact of the gut microbiota on pulmonary responses to ozone. After acute exposure to ozone, male mice developed greater airway hyperresponsiveness than female mice. This difference was abolished after antibiotic ablation of the gut microbiome. Moreover, weanling female pups housed in cages conditioned by adult male mice developed greater ozone-induced airway hyperresponsiveness than weanling female pups raised in cages conditioned by adult females. Finally, ad libitum oral administration via drinking water of the short-chain fatty acid propionate resulted in augmented ozone-induced airway hyperresponsiveness in male, but not female, mice. Overall, these data are consistent with the hypothesis that the microbiome contributes to sex differences in ozone-induced airway hyperresponsiveness, likely as a result of sex differences in the response to short-chain fatty acids.