Factor-based assessment of continuous bio-H2 production from cheese whey.

Despite having been widely investigated, dark fermentative H2 production from organic residues is still limited by process-related issues which may hamper the perspectives of full-scale process implementation. Such constraints are mainly due to the process complexity, which is largely affected by multiple and often mutually interacting factors. In the present work, the results of continuous fermentative H2 production experiments using synthetic cheese whey as the input substrate were used to gain detailed knowledge of the process features and identify suitable and critical operating conditions. Specifically, innovative process interpretation involved a combination of analytical characterization of the fermentation broth, mass balance calculations and statistical methods (correlation and principal component analyses) to derive systematic considerations for process characterization and scale-up. The metabolic products mainly included acetate and butyrate, which however were likely to derive (in different proportions depending on the operating conditions) from both hydrogenogenic and competing pathways. For some tests, lactate and succinate were also found to have been formed. It was observed that the main features of the process (H2 yield and rate, stability condition) were correlated with the operational and analytical parameters. The first three principal components identified by the statistical analysis were able to account for: 1) the effect of retention time and total metabolites produced; 2) biogas (H2 and CO2) generation, butyrate production and stability condition; and 3) organic loading rate and propionate production. The results suggested that the main features of hydrogenogenic fermentation can be described by a reduced set of factors that may be usefully adopted for both process monitoring and prediction purposes.

Production, Structural Characterization, and In Vitro Assessment of the Prebiotic Potential of Butyl-Fructooligosaccharides.

Short-chain fatty acids (SCFAs), especially butyrate, produced in mammalian intestinal tracts via fermentation of dietary fiber, are known biofunctional compounds in humans. However, the variability of fermentable fiber consumed on a daily basis and the diversity of gut microbiota within individuals often limits the production of short-chain fatty acids in the human gut. In this study, we attempted to enhance the butyrate levels in human fecal samples by utilizing butyl-fructooligosaccharides (B-FOS) as a novel prebiotic substance. Two major types of B-FOS (GF3-1B and GF3-2B), composed of short-chain fructooligosaccharides (FOS) bound to one or two butyric groups by ester bonds, were synthesized. Qualitative analysis of these B-FOS using Fourier transform infrared (FT-IR) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), nuclear magnetic resonance (NMR) and low-resolution fast-atom bombardment mass spectra (LR-FAB-MS), showed that the chemical structure of GF3-1B and GF3-2B were [O-(1-buty-ß-D-fru-(2→1)-O-ß-D-fru-(2→1)-O-ß-D-fru-O-α-D-glu] and [O-(1-buty)-ß-D-fru-(2→1)-O-ß-D-fru-(2→1)-O-(4-buty)-ß-D-fru-O-α-D-glu], respectively. The ratio of these two compounds was approximately 5:3. To verify their biofunctionality as prebiotic oligosaccharides, proliferation and survival patterns of human fecal microbiota were examined in vitro via 16S rRNA metagenomics analysis compared to a positive FOS control and a negative control without a carbon source. B-FOS treatment showed different enrichment patterns on the fecal microbiota community during fermentation, and especially stimulated the growth of major butyrate producing bacterial consortia and modulated specific butyrate producing pathways with significantly enhanced butyrate levels. Furthermore, the relative abundance of Fusobacterium and ammonia production with related metabolic genes were greatly reduced with B-FOS and FOS treatment compared to the control group. These findings indicate that B-FOS differentially promotes butyrate production through the enhancement of butyrate-producing bacteria and their metabolic genes, and can be applied as a novel prebiotic compound in vivo.

Genomic assessment in Lactobacillus plantarum links the butyrogenic pathway with glutamine metabolism.

The butyrogenic capability of Lactobacillus (L.) plantarum is highly dependent on the substrate type and so far not assigned to any specific metabolic pathway. Accordingly, we compared three genomes of L. plantarum that showed a strain-specific capability to produce butyric acid in human cells growth media. Based on the genomic analysis, butyric acid production was attributed to the complementary activities of a medium-chain thioesterase and the fatty acid synthase of type two (FASII). However, the genomic islands of discrepancy observed between butyrogenic L. plantarum strains (S2T10D, S11T3E) and the non-butyrogenic strain O2T60C do not encompass genes of FASII, but several cassettes of genes related to sugar metabolism, bacteriocins, prophages and surface proteins. Interestingly, single amino acid substitutions predicted from SNPs analysis have highlighted deleterious mutations in key genes of glutamine metabolism in L. plantarum O2T60C, which corroborated well with the metabolic deficiency suffered by O2T60C in high-glutamine growth media and its consequent incapability to produce butyrate. In parallel, the increase of glutamine content induced the production of butyric acid by L. plantarum S2T10D. The present study reveals a previously undescribed metabolic route for butyric acid production in L. plantarum, and a potential involvement of the glutamine uptake in its regulation.

Probiotic Lactobacillus gasseri SBT2055 improves glucose tolerance and reduces body weight gain in rats by stimulating energy expenditure.

Probiotic Lactobacillus gasseri SBT2055 (LG2055) reduces postprandial TAG absorption and exerts anti-obesity effects in rats and humans; however, the underlying mechanisms are not fully understood. In the present study, we addressed the mechanistic insights of the anti-obesity activity of LG2055 by feeding Sprague-Dawley rats diets containing skimmed milk fermented or not by LG2055 for 4 weeks and by analysing energy expenditure, glucose tolerance, the levels of SCFA in the caecum and serum inflammatory markers. Rats fed the LG2055-containing diet demonstrated significantly higher carbohydrate oxidation in the dark cycle (active phase for rats) compared with the control group, which resulted in a significant increase in energy expenditure. LG2055 significantly reduced cumulative blood glucose levels (AUC) compared with the control diet after 3 weeks and increased the molar ratio of butyrate:total SCFA in the caecum after 4 weeks. Furthermore, the LG2055-supplemented diet significantly reduced the levels of serum amyloid P component - an indicator of the inflammatory process. In conclusion, our results demonstrate that, in addition to the inhibition of dietary TAG absorption reported previously, the intake of probiotic LG2055 enhanced energy expenditure via carbohydrate oxidation, improved glucose tolerance and attenuated inflammation, suggesting multiple additive and/or synergistic actions underlying the anti-obesity effects exerted by LG2055.

A novel live cell assay to measure diacylglycerol lipase α activity.

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

Safety assessment of the butyrate-producing Butyricicoccus pullicaecorum strain 25-3(T), a potential probiotic for patients with inflammatory bowel disease, based on oral toxicity tests and whole genome sequencing.

Inflammatory bowel disease (IBD) is a chronic inflammation of the digestive tract, characterized by dysbiosis of the intestinal microbiota. Probiotics have been suggested as a strategy to reduce active disease or extend remission. We isolated and characterized the butyrate-producing strain Butyricicoccus pullicaecorum 25-3(T) and identified it as a potential probiotic for patients with IBD. To evaluate the safety of 25-3(T) for use in humans, we conducted a standard acute oral toxicity test and a 28-day repeated oral dose toxicity test. The complete genome of B. pullicaecorum 25-3(T) was sequenced to search for virulence factors and antibiotic resistance determinants. The minimum inhibitory concentration (MIC) of 21 antimicrobials was determined. Results showed no adverse effects in the oral toxicity tests. B. pullicaecorum 25-3(T) is resistant against aminoglycosides and trimethoprim. The genome of 25-3(T) contains no virulence factors, one gene related to harmful metabolites and 52 sequences with high similarity to antimicrobial and toxic compound resistance genes, that did not correspond with a resistant phenotype. This first report of a safety assessment of a butyrate-producing strain from Clostridium cluster IV shows that B. pullicaecorum 25-3(T) is a non-pathogenic strain, but carries antibiotic resistance genes with the risk of transfer, that need further investigation.

Butyrate-producing Clostridium cluster XIVa species specifically colonize mucins in an in vitro gut model.

The human gut is colonized by a complex microbiota with multiple benefits. Although the surface-attached, mucosal microbiota has a unique composition and potential to influence human health, it remains difficult to study in vivo. Therefore, we performed an in-depth microbial characterization (human intestinal tract chip (HITChip)) of a recently developed dynamic in vitro gut model, which simulates both luminal and mucosal gut microbes (mucosal-simulator of human intestinal microbial ecosystem (M-SHIME)). Inter-individual differences among human subjects were confirmed and microbial patterns unique for each individual were preserved in vitro. Furthermore, in correspondence with in vivo studies, Bacteroidetes and Proteobacteria were enriched in the luminal content while Firmicutes rather colonized the mucin layer, with Clostridium cluster XIVa accounting for almost 60% of the mucin-adhered microbiota. Of the many acetate and/or lactate-converting butyrate producers within this cluster, Roseburia intestinalis and Eubacterium rectale most specifically colonized mucins. These 16S rRNA gene-based results were confirmed at a functional level as butyryl-CoA:acetate-CoA transferase gene sequences belonged to different species in the luminal as opposed to the mucin-adhered microbiota, with Roseburia species governing the mucosal butyrate production. Correspondingly, the simulated mucosal environment induced a shift from acetate towards butyrate. As not only inter-individual differences were preserved but also because compared with conventional models, washout of relevant mucin-adhered microbes was avoided, simulating the mucosal gut microbiota represents a breakthrough in modeling and mechanistically studying the human intestinal microbiome in health and disease. Finally, as mucosal butyrate producers produce butyrate close to the epithelium, they may enhance butyrate bioavailability, which could be useful in treating diseases, such as inflammatory bowel disease.

Continuous lactose fermentation by Clostridium acetobutylicum--assessment of energetics and product yields of the acidogenesis.

An assessment of both the growth and the metabolism of acidogenic cells Clostridium acetobutylicum DSM 792 is reported in the paper. Tests were carried out in a CSTR under controlled pH conditions. Cultures were carried out using a semi-synthetic medium supplemented with lactose as carbon source. Acids and solvents, that represent products of the ABE process, have been purposely added in controlled amounts to the culture medium to investigate their effects on the product yields. The mass fractional yield of biomass and products were expressed as a function of the specific growth rate taking into account the Pirt model. The maximum ATP yield and the maintenance resulted 29.1 g(DM)/mol(ATP) and 0.012 mol(ATP)/g(DM)h, respectively. Quantitative features of the C. acetobutylicum growth model were in good agreement with experimental results. The model proposes as a tool to estimate the mass fractional yield even for fermentations carried out under conditions typical of the solventogenesis.

Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli.

In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at much lower titers and yields. To pinpoint deficient steps in the recombinant pathway, we developed an analytical procedure for the determination of intracellular pools of key pathway intermediates and applied the technique to the analysis of three sets of E. coli strains expressing various combinations of butyrate biosynthesis genes. Low expression levels of the hbd-encoded S-3-hydroxybutyryl-CoA dehydrogenase were insufficient to convert acetyl-CoA to 3-hydroxybutyryl-CoA, indicating that hbd was a rate-limiting step in the production of butyryl-CoA. Increasing hbd expression alleviated this bottleneck, but in resulting strains, our pool size measurements and thermodynamic analysis showed that the reaction step catalyzed by the bcd-encoded butyryl-CoA dehydrogenase was rate-limiting. E. coli strains expressing both hbd and ptb-buk produced crotonic acid as a byproduct, but this byproduct was not observed with expression of related genes from non-clostridial organisms. Our thermodynamic interpretation of pool size measurements is applicable to the analysis of other metabolic pathways.

Simultaneous wastewater treatment and biological electricity generation.

It is possible to directly generate electricity using bacteria while accomplishing wastewater treatment in processes based on microbial fuel cell technologies. When bacteria oxidize a substrate, they remove electrons. Current generation is made possible by keeping bacteria separated from oxygen, but allowing the bacteria growing on an anode to transfer electrons to the counter electrode (cathode) that is exposed to air. In this paper, several advances are discussed in this technology, and a calculation is made on the potential for electricity recovery. Assuming a town of 100,000 people generate 16.4 x 10(6) L of wastewater, a wastewater treatment plant has the potential to become a 2.3 MW power plant if all the energy is recovered as electricity. So far, power densities are low, resulting in power generation rates of 150 kW/m2. Progress is being made that we believe may result in as much as 0.5 MW from wastewater treatment. The generation of electricity during wastewater treatment may profoundly affect the approach to anaerobic treatment technologies used in wastewater treatment.

Evaluation of a sunscreen photoprotective effect by ascorbic acid assessment in human dermis using microdialysis and gas chromatography mass spectrometry.

Ultraviolet irradiation causes adverse effects like sunburn, photosensitivity reactions or immunologic suppression. The aim of this study was to evaluate the photo-protective outcome of a sunscreen cream (SPF8) by the determination of erythema indexes and the assessment of ascorbic acid and its metabolites in human dermis. These substances were used as markers of oxidative effect. Eight healthy female subjects were enrolled in this study. Two abdominal areas were exposed to solar simulated irradiation with three minimal erythema dose, one with SPF8 application and the other site without SPF8 application. Two other areas were used as control, one without SPF8 application and the other site after SPF8 application. Ascorbic acid and its metabolites (dehydroascorbic acid, threonic acid, oxalic acid and xylose) were collected from human dermis by microdialysis and assessed by gas chromatography mass spectrometry. Irradiated site without sunscreen application had significantly demonstrated lower dermis ascorbic acid concentrations and a higher erythema index than the three other sites (P < 0.05). Threonic acid, oxalic acid and xylose dermis concentrations were significantly higher in site III than in the control site I (P < 0.05). The protected-irradiated site did not show erythema formation and there was stability of ascorbic acid dermis concentrations with non-variation in its metabolites. The assessment of ascorbic acid and its metabolites in human dermis could be an efficient tool to demonstrate the oxidative process and consequently to control the efficiency of sunscreen creams against undesirable UV effects.

A simple method to establish short-term cultures of normal human colonic epithelial cells from endoscopic biopsy specimens. Comparison of isolation methods, assessment of viability and metabolic activity.

BACKGROUND: Abnormalities in colonic epithelial cell function have been implicated in the pathogenesis of various intestinal disorders, especially inflammatory bowel disease (IBD). The mechanisms, however, remain obscure owing to the lack of representative human colonic epithelial cell models. The aim of this study was to develop and validate a method for establishment of short-term culture of normal human colonic epithelial cells from endoscopic biopsies. METHODS: Epithelial cells were isolated from colonoscopic biopsies by means of ethylenediaminetetraacetic acid/ethylene glycol tetraacetic acid (EDTA/EGTA) (10 or 60 min) or by enzyme treatment and cultured in collagen-coated wells. Viability was measured with a methyltetrazoleum conversion assay, confocal laser, and electron microscopy. Metabolic function was measured by means of butyrate oxidation, 14C-leucine and 3H-glucosamine incorporation; DNA synthesis by means of 3H-thymidine incorporation, and apoptosis with an enzyme-linked immunosorbent assay (ELISA) for histone-associated DNA fragments. Cell types were identified by immunocytochemistry. RESULTS: Ten minutes of EDTA/EGTA treatment released intact crypts and was superior to both the 60-min treatment and enzymatic treatment in terms of viability and nonepithelial cell contamination, respectively. Despite activation of detachment-induced apoptosis, a median 51% of the isolated cells was viable after 24 h of culture and metabolically active as judged by 3H-thymidine, 14C-leucine, and 3H-glucosamine incorporation. Butyrate oxidation followed more complex kinetics (substrate activation) than observed previously in other models. The apparent Km values (medians) were 0.7 mM and 4.5 mM in low and high concentration ranges, respectively. CONCLUSION: We report a simple method to establish culture of human colonic epithelial cells from endoscopically obtained biopsy specimens, producing sufficient viable cells to perform metabolic studies pertinent to the pathogenesis of IBD and related human disorders.

Evaluation of diets containing supplemental fat with different sources of carbohydrates for lactating dairy cows.

A study was conducted to evaluate the lactational response of high producing cows to diets supplemented with fat that contained additional ruminally degradable carbohydrate from a molasses plus fat product and dried whey. Forty Holstein cows were randomly assigned within lactation group to receive diets containing 2% tallow with or without molasses or dried whey wk 4 through 16 postpartum. Cows were fed 1) the control TMR of 25% corn silage, 25% alfalfa hay, and 50% concentrate mix, 2) the TMR containing fat, 3) the TMR containing molasses and fat, or 4) the TMR containing dried whey and fat. Production of milk and 3.5% FCM was increased by supplemental fat. Milk protein and fat percentages were not affected by supplemental fat with or without molasses or dried whey. The DMI and BW were similar for all diets. Production efficiency (3.5% FCM/DMI) was higher for cows fed supplemental fat diets, and cows fed tallow alone were more efficient than those fed tallow with molasses or dried whey. Tallow did not influence ruminal concentrations of various VFA. Molar percentage of butyrate was higher for cows fed the TMR containing molasses plus fat or dried whey plus fat than for cows fed the TMR containing fat. The TMR containing 2% tallow increased milk production, but no economic advantage was derived from inclusion of an additional ruminally fermentable carbohydrate as molasses or from dried whey with fat.

A quantitative assessment of F-actin content and distribution in untreated and butyric acid treated murine melanoma B16a tumour cells: a fluorescence image analysis study.

Image analysis of phallacidin, a fluorescent stoichiometric probe to F-actin, permitted the cytoskeletal-associated actin 'F-actin' to be visualized morphologically and to be divided into two groups, diffuse and filamentous. The filamentous actin group was categorized further into four subgroups according to the intensity of the phallacidin probe. F-actin groups and subgroups of untreated cells and cells treated with 1.5 mM butyrate acid were analysed independently. Butyric acid treatment significantly increased total actin, defined as diffuse actin, plus filamentous subgroup actins 1-4. Specifically, butyric acid-treatment increased filamentous subgroup actin 1.