Fermentation End Products Research Articles

Purification and fermentation characteristics of exopolysaccharide from Fomitopsis castaneus Imaz

Short-chain fatty acids (SCFAs), which are the end products of carbohydrate fermentation in the gut, mainly contribute to energy metabolism in mammals. The amount of SCFAs produced during fermentation is an important parameter that characterizes the fermentation capacity of a system. This paper reports on the fermentation characteristics of exopolysaccharides (EPS) isolated from Fomitopsis castaneus Imaz, a wood-rot fungal species. We isolated and purified the main EPS fraction by freeze drying and DEAE-Sepharose fast flow chromatography. We then analyzed the monosaccharide composition of EPS. The isolated EPS was mainly composed of glucose, galactose, rhamnose, mannose, and arabinose. The characteristic absorption peaks of sugar esters were also detected. Fresh fecal extracts from healthy adults and children were used as fermentation substrate to simulate the human intestinal environment (anaerobic conditions at 37°C) and study the fermentation characteristics of the purified EPS. Adding the isolated EPS to the fermentation system of the simulated intestinal environment increased the SCFAs content in the fecal extract of adults and children. However, the yield of SCFAs, particularly butyric acid, in the fermentation system of fecal extract in children was higher than that in adults. Furthermore, adding exogenous lactic acid bacteria, such as Enterococcus fecalis and Enterococcus fecium, to the fermentation system effectively increased the SCFAs concentration in the model intestinal system of the children. By contrast, adding E. fecalis, Lactobacillus rhamnosus, and E. fecium increased the content of the produced SCFAs in the system of adults. Those results indicate that EPS isolated from F. castaneus Imaz was effectively fermented in the simulated intestinal environments, and the fermentation capability was enhanced by adding microbial flora.

Effect of Sunflower and Marine Oils on Ruminal Microbiota, In vitro Fermentation and Digesta Fatty Acid Profile.

This study using the rumen simulation technique (RUSITEC) investigated the changes in the ruminal microbiota and anaerobic fermentation in response to the addition of different lipid supplements to a ruminant diet. A basal diet with no oil added was the control, and the treatment diets were supplemented with sunflower oil (2%) only, or sunflower oil (2%) in combination with fish oil (1%) or algae oil (1%). Four fermentation units were used per treatment. RUSITEC fermenters were inoculated with rumen digesta. Substrate degradation, fermentation end-products (volatile fatty acids, lactate, gas, methane, and ammonia), and microbial protein synthesis were determined. Fatty acid profiles and microbial community composition were evaluated in digesta samples. Numbers of representative bacterial species and microbial groups were determined using qPCR. Microbial composition and diversity were based on T-RFLP spectra. The addition of oils had no effect on substrate degradation or microbial protein synthesis. Differences among diets in neutral detergent fiber degradation were not significant (P = 0.132), but the contrast comparing oil–supplemented diets with the control was significant (P = 0.039). Methane production was reduced (P < 0.05) with all oil supplements. Propionate production was increased when diets containing oil were fermented. Compared with the control, the addition of algae oil decreased the percentage C18:3 c9c12c15 in rumen digesta, and that of C18:2 c9t11 was increased when the control diet was supplemented with any oil. Marine oils decreased the hydrogenation of C18 unsaturated fatty acids. Microbial diversity was not affected by oil supplementation. Cluster analysis showed that diets with additional fish or algae oils formed a group separated from the sunflower oil diet. Supplementation with marine oils decreased the numbers of Butyrivibrio producers of stearic acid, and affected the numbers of protozoa, methanogens, Selenomonas ruminantium and Streptococcus bovis, but not total bacteria. In conclusion, there is a potential to manipulate the rumen fermentation and microbiota with the addition of sunflower, fish or algae oils to ruminant diets at appropriate concentrations. Specifically, supplementation of ruminant mixed rations with marine oils will reduce methane production, the acetate to propionate ratio and the fatty acid hydrogenation in the rumen.

Nutritive value of corn silage from intensive dairy farms in Brazil

ABSTRACT The objective of this research was to characterize the nutritive value of corn silage made on intensive dairy farms and demonstrate the nutritional variations between silages located at the top and at the center of a bunker silo. Thirty-two dairy farms were visited in four Brazilian states. One corn bunker silo of each farm was chosen and samples were collected from the top and center parts. The nutritive value, fermentation end-products, and microbial counts were assessed. The predicted milk [...]

Performance of a pilot split-type anaerobic membrane bioreactor (AnMBR) treating antibiotics solvent wastewater at low temperatures

The performance of a new pilot split-type anaerobic membrane bioreactor (AnMBR) configuration for treating antibiotics solvent wastewater at low temperatures was evaluated in this study. The AnMBR system comprised an internal circulation (IC) and ultrafiltration membrane modules, working as the biological removal and physical retention, respectively. The whole process was divided into four stages in terms of the variation of temperatures (35±3°C, 25±3°C, 15±3°C, 25±3°C). Total chemical oxygen demand (CODtotal) and tetrahydrofuran (THFtotal) removal efficiency at temperatures as low as 15±3°C were similar to mesophilic conditions (35±3°C, 25±3°C), which achieved over 95% and 96%, respectively. While different mechanism of removal was obtained at different temperatures. The excellent removal efficiency was maintained via the combination of biological and physical removal, biological removal was the major reason of removal efficiency (>83.3%) at mesophilic temperatures and physical removal (>38.6%) dominated the effect at lower temperatures. As temperature decreased, permeate volatile fatty acids (VFA) in the bioreactor accumulated suggesting a reduction of methanogens activity and pH had a minimum value of 6.5. However, neither of them showed significant sign of water degradation towards the end of experiment. The examination of alkalinity, α value, and end-products of anaerobic fermentation, MLSS, MLVSS and MLVSS/MLSS suggested a negative effect slightly on microbial activity and no negative effect on the effluent quality at low temperatures due to the retention of membrane.

Utilizing Anaerobic Fungi for Two-stage Sugar Extraction and Biofuel Production from Lignocellulosic Biomass.

Lignocellulosic biomass is a vast and underutilized resource for the production of sugars and biofuels. However, the structural complexity of lignocellulosic biomass and the need for multiple pretreatment and enzymatic steps for sugar release renders this process economically challenging. Here, we report a novel approach for direct, single container, exogenous enzyme-free conversion of lignocellulosic biomass to sugars and biofuels using the anaerobic fungal isolate strain C1A. This approach utilizes simple physiological manipulations for timely inhibition and uncoupling of saccharolytic and fermentative capabilities of strain C1A, leading to the accumulation of sugar monomers (glucose and xylose) in the culture medium. The produced sugars, in addition to fungal hyphal lysate, are subsequently converted by Escherichia coli strain K011 to ethanol. Using this approach, we successfully recovered 17.0% (w/w) of alkali-pretreated corn stover (20.0% of its glucan and xylan content) as sugar monomers in the culture media. More importantly, 14.1% of pretreated corn stover (17.1% of glucan and xylan content) was recovered as ethanol at a final concentration of 28.16 mM after the addition of the ethanologenic strain K011. The high ethanol yield obtained is due to its accumulation as a minor fermentation end product by strain C1A during its initial growth phase, the complete conversion of sugars to ethanol by strain K011, and the possible conversion of unspecified substrates in the hyphal lysate of strain C1A to ethanol by strain K011. This study presents a novel, versatile, and exogenous enzyme-free strategy that utilizes a relatively unexplored group of organisms (anaerobic fungi) for direct biofuel production from lignocellulosic biomass.

Prebiotic evaluation of red seaweed (Kappaphycus alvarezii) using in vitro colon model

Red seaweed (Kappaphycus alvarezii) cultivated from Sabah (RSS) and Langkawi (RSL) were digested using in vitro mouth, gastric and duodenal model. The digested seaweed then fermented in a pH-controlled batch culture system inoculated with human faeces to mimic the distal colon. Bacterial enumeration were monitored using fluorescent in situ hybridisation, and the fermentation end products, the short chain fatty acids (SCFA), were analysed using HPLC. Both RSS and RSL showed significant increase of Bifidobacterium sp.; from log10 7.96 at 0 h to log10 8.72 at 24 h, and from log10 7.96 at 0 h to log10 8.60 at 24 h, respectively, and shows no significant difference when compared to the Bifidobacterium sp. count at 24 h of inulin fermentation. Both seaweeds also showed significant increase in total SCFA production, particularly acetate and propionate. Overall, this data suggested that K. alvarezii might have the potential as a prebiotic ingredient.

Supplementation of Diet With Galacto-oligosaccharides Increases Bifidobacteria, but Not Insulin Sensitivity, in Obese Prediabetic Individuals

The gut microbiota affects host lipid and glucose metabolism, satiety, and chronic low-grade inflammation to contribute to obesity and type 2 diabetes. Fermentation end products, in particular the short-chain fatty acid (SCFA) acetate, are believed to be involved in these processes. We investigated the long-term effects of supplementation with galacto-oligosaccharides (GOS), an acetogenic fiber, on the composition of the human gut microbiota and human metabolism. We performed a double-blinded, placebo-controlled, parallel intervention study of 44 overweight or obese (body mass index, 28-40 kg/m2) prediabetic men and women (ages, 45-70 y) from October 2014 through October 2015 in Maastricht, The Netherlands. The participants were assigned randomly to groups who ingested 15 g GOS or isocaloric placebo (maltodextrin) daily with their regular meals for 12 weeks. Before and after this period, we collected data on peripheral and adipose tissue insulin sensitivity, fecal microbiota composition, plasma and fecal SCFA, energy expenditure and substrate oxidation, body composition, and hormonal and inflammatory responses. The primary outcome was the effect of GOS on peripheral insulin sensitivity, measured by the hyperinsulinemic-euglycemic clamp method. Supplementation of diets with GOS, but not placebo, increased the abundance of Bifidobacterium species in feces by 5-fold (P= .009; q= 0.144). Microbial richness or diversity in fecal samples were not affected. We did not observe any differences in fecal or fasting plasma SCFA concentrations or in systemic concentrations of gut-derived hormones, incretins, lipopolysaccharide-binding protein, or other markers of inflammation. In addition, no significant alterations in peripheral and adipose tissue insulin sensitivity, body composition, and energy and substrate metabolism were found. Twelve-week supplementation of GOS selectively increased fecal Bifidobacterium species abundance, but this did not produce significant changes in insulin sensitivity or related substrate and energy metabolism in overweight or obese prediabetic men and women. ClincialTrials.gov number, NCT02271776.

Lining bunker walls with oxygen barrier film reduces nutrient losses in corn silages

The objective of this study was to evaluate 2 systems for covering corn silage in bunker silos. The first system consisted of a sheet of 45-μm-thick oxygen barrier film (OB, polyethylene + ethylene-vinyl alcohol) placed along the length of the sidewall before filling. After filling, the excess film was pulled over the wall on top of the silage, and a sheet of polyethylene was placed on top. The second system involved using a standard sheet (ST) of 180-μm-thick polyethylene film. Eight commercial bunker silos were divided into 2 parts lengthwise so that one-half of the silo was covered with OB and the other half with a ST system. During the filling, 3 net bags with chopped corn were buried in the central part (halfway between the top and bottom of the silo) of the bunkers (CCOR) in 3 sections 10 m apart. After filling, 18 net bags (9 per covering system) were buried 40 cm below the top surface of the 3 sections. These bags were placed at 3 distances from the bunker walls (0 to 50 cm, 51 to 100 cm, and 101 to 150 cm). During unloading, the bags were removed from the silos to determine the dry matter (DM) losses, fermentation end products, and nutritive value. The Milk2006 spreadsheet was used to estimate milk per tonne of DM. The model included the fixed effect of treatment (7 different locations in the bunker) and the random effect of the silo. Two contrasts were tested to compare silages in the top laterals (shoulders) with that in the CCOR (CCOR vs. OB and CCOR vs. ST). Three contrasts compared the corresponding distances of the silage covered by the 2 systems (OB50 vs. ST50, OB100 vs. ST100 and OB150 vs. ST150). Variables were analyzed with the PROC MIXED procedure of the SAS at the 5% level. The OB method produced well-fermented silages, which were similar to CCOR, whereas the OB system showed less lactic acid and greater pH and mold counts compared with CCOR. The ST method had 116.2 kg of milk/t less than the CCOR, as the OB system and the CCOR were similar (1,258.3 and 1,294.0 kg/t, respectively). Regarding the distances from the walls, the effects were more pronounced from 0 to 101 cm. The OB50 and OB100 silages had better quality and lower mold counts and DM losses than ST50 and ST100. The OB system reduced DM and nutrient losses at the shoulders in farm bunker corn silages compared with no sidewall plastic. The OB film should lap onto the crop for at least 200 cm so that 150 cm are covered outward from the wall.

Microbial Metabolite, Butyrate, Increases Blood Pressure in Rodent Models of Hypertension

IntroductionThe emerging link between gut dysbiosis and hypertension is characterized by a significant reduction in butyrate‐producing bacteria in the spontaneously hypertensive rat (SHR) compared with the Wistar Kyoto (WKY) control. Butyrate, a major end product of dietary fiber fermentation by gut microbiota, reportedly produces beneficial effects in multiple dysbiosis‐related diseases. However, butyrate has also been linked with activation of the sympathetic drive, elevated heart rate and release of norepinephrine. Considering the established link between sympathetic drive and hypertension, we investigated the role of butyrate in blood pressure (BP) control in hypertension.MethodsMale SHR (5 wo) were placed on either the fiber‐rich (butyrolytic) diet (SHR‐BD) or control diet (SHR‐CD) for 13 weeks ad libitum. Fecal/cecal butyrate levels were determined using high performance liquid chromatography (HPLC) throughout the study. Following this, fecal matter transplant (FMT) from both groups was performed separately to male WKY recipients (7 wo) with antibiotic‐depleted endogenous gut bacteria, to create two groups: WKY FMT‐BD and WKY FMT‐CD. Five weeks following FMT, both groups were infused with a subpressor dose of angiotensin II (Ang II; 50ng/kg/min) for three weeks. Separate male SHR (9 wo) were administered with daily injections of either saline or butyrate (500mg/kg/day I.P.) for four weeks. BPs were measured in all groups using tailcuff or telemetry throughout the study.ResultsElevated cecal butyrate in SHR‐BD (by 2 fold vs. SHR‐CD; P&lt;0.001) was associated with significantly higher SBP (SHR‐CD: 183.3mmHg vs SHR‐BD: 206.6 mmHg; P&lt;0.01). FMT from SHR‐CD and SHR‐BD caused a similar BP increase in the WKY recipients (ΔSBP=11.5 mmHg, P&lt;0.001), However, a subsequent administration of subpressor Ang II further elevated SBP in the WKY FMT‐BD group only (by 7.7 mmHg vs. WKY FMT‐CD; P=0.06). Finally, chronic IP injection of butyrate in the SHR resulted in significant SBP increase compared to saline‐injected SHR (ΔSBP=14.5 mmHg, P&lt;0.05).ConclusionSHR gut microbiota is pro‐hypertensive in the WKY. Moreover, subpressor Ang II exacerbates this hypertension in the presence of excess butyrate. Thus, elevated butyrate may interact with Ang II in the pathophysiology of hypertension, indicating the disadvantageous role of butyrate in the context of hypertension.Support or Funding Information1. American Heart Association 2. College of Veterinary Medicine, University of Florida

Methane Production in Dairy Cows Correlates with Rumen Methanogenic and Bacterial Community Structure.

Methane (CH4) is produced as an end product from feed fermentation in the rumen. Yield of CH4 varies between individuals despite identical feeding conditions. To get a better understanding of factors behind the individual variation, 73 dairy cows given the same feed but differing in CH4 emissions were investigated with focus on fiber digestion, fermentation end products and bacterial and archaeal composition. In total 21 cows (12 Holstein, 9 Swedish Red) identified as persistent low, medium or high CH4 emitters over a 3 month period were furthermore chosen for analysis of microbial community structure in rumen fluid. This was assessed by sequencing the V4 region of 16S rRNA gene and by quantitative qPCR of targeted Methanobrevibacter groups. The results showed a positive correlation between low CH4 emitters and higher abundance of Methanobrevibacter ruminantium clade. Principal coordinate analysis (PCoA) on operational taxonomic unit (OTU) level of bacteria showed two distinct clusters (P < 0.01) that were related to CH4 production. One cluster was associated with low CH4 production (referred to as cluster L) whereas the other cluster was associated with high CH4 production (cluster H) and the medium emitters occurred in both clusters. The differences between clusters were primarily linked to differential abundances of certain OTUs belonging to Prevotella. Moreover, several OTUs belonging to the family Succinivibrionaceae were dominant in samples belonging to cluster L. Fermentation pattern of volatile fatty acids showed that proportion of propionate was higher in cluster L, while proportion of butyrate was higher in cluster H. No difference was found in milk production or organic matter digestibility between cows. Cows in cluster L had lower CH4/kg energy corrected milk (ECM) compared to cows in cluster H, 8.3 compared to 9.7 g CH4/kg ECM, showing that low CH4 cows utilized the feed more efficient for milk production which might indicate a more efficient microbial population or host genetic differences that is reflected in bacterial and archaeal (or methanogens) populations.

Butyrate induces apoptosis by activating PDC and inhibiting complex I through SIRT3 inactivation

The underlying anticancer effects of butyrate, an end-product of the intestinal microbial fermentation of dietary fiber, remain elusive. Here, we report that butyrate promotes cancer cell apoptosis by acting as a SIRT3 inhibitor. Butyrate inhibits SIRT3 both in cultured cells and in vitro. Butyrate-induced PDHA1 hyperacetylation relieves the inhibitory phosphorylation of PDHA1 at serine 293, thereby activating an influx of glycolytic intermediates into the tricarboxylic acid (TCA) cycle and reversing the Warburg effect. Meanwhile, butyrate-induced hyperacetylation inactivates complex I of the electron transfer chain and prevents the utilization of TCA cycle intermediates. These metabolic stresses promote apoptosis in hyperglycolytic cancer cells, such as HCT116p53−/− cells. SIRT3 deacetylates both PDHA1 and complex I. Genetic ablation of Sirt3 in mouse hepatocytes abrogated the ability of butyrate to induce apoptosis. Our results identify a butyrate-mediated anti-tumor mechanism and indicate that the combined activation of PDC and inhibition of complex I is a novel tumor treatment strategy.

Fermentation of model hemicelluloses by Prevotella strains and Butyrivibrio fibrisolvens in pure culture and in ruminal enrichment cultures.

Hemicelluloses are major components of plant biomass, but their fermentation in the rumens of cattle and other ruminants is poorly understood. We compared four species of the ruminally dominant genus Prevotella and the well-known hemicellulose utilizer, Butyrivibrio fibrisolvens, with respect to degradation of several isolated hemicelluloses (xylans, glucomannan, and xyloglucan). We also performed Illumina sequencing of the V3/V4 region of 16S rRNA genes to determine the relative proportions of Prevotella and Butyrivibrio in hemicellulose-fed enrichment cultures inoculated from ruminal contents of dairy cattle fed a total mixed ration (TMR) rich in hemicelluloses. Results confirmed the xylan fermentation and butyrate production abilities of B. fibrisolvens. Despite their reputation as generalist fermenters, the Prevotella strains poorly fermented these hemicelluloses but exhibited dramatic differences in fermentation end products. Prevotella was much less abundant in mixed bacterial enrichment cultures fed the same TMR than in the ruminal inoculum, yet Prevotella was again the most abundant genus in enrichment cultures fed xylans. By contrast, glucomannan fermentations were dominated by Streptococcus sp. Genera known for hemicellulose degradation (Butyrivibrio, Ruminococcus, and Fibrobacter) were not significantly enriched on these hemicelluloses. Substantial differences in fermentation end product distribution from the different hemicelluloses were observed, which would likely affect nutrient partitioning in the host animal. Differences in community composition between in vitro hemicellulose enrichments and inoculum samples emerged at every phylogenetic level, suggesting that in vitro conditions provide unique selective pressures on the bacterial community and also that ruminal bacteria exhibit specialization with respect to hemicellulose utilization.

Differences in Gut Metabolites and Microbial Composition and Functions between Egyptian and U.S. Children Are Consistent with Their Diets.

Previous studies indicated that populations consuming a Mediterranean diet rich in fiber, vegetables, and fruits have a significantly lower risk of cardiovascular and metabolic diseases than populations of industrialized societies consuming diets enriched in processed carbohydrates, animal proteins, and fats. To explore the potential contributions of gut microbiota to the observed diet-related metabolic effects, we conducted an integrative analysis of distal gut microbiota composition and functions and intestinal metabolites in Egyptian and U.S. teenagers. All Egyptian gut microbial communities belonged to the Prevotella enterotype, whereas all but one of the U.S. samples were of the Bacteroides enterotype. The intestinal environment of Egyptians was characterized by higher levels of short-chain fatty acids, a higher prevalence of microbial polysaccharide degradation-encoding genes, and a higher proportion of several polysaccharide-degrading genera. Egyptian gut microbiota also appeared to be under heavier bacteriophage pressure. In contrast, the gut environment of U.S. children was rich in amino acids and lipid metabolism-associated compounds; contained more microbial genes encoding protein degradation, vitamin biosynthesis, and iron acquisition pathways; and was enriched in several protein- and starch-degrading genera. Levels of 1-methylhistamine, a biomarker of allergic response, were elevated in U.S. guts, as were the abundances of members of Faecalibacterium and Akkermansia, two genera with recognized anti-inflammatory effects. The revealed corroborating differences in fecal microbiota structure and functions and metabolite profiles between Egyptian and U.S. teenagers are consistent with the nutrient variation between Mediterranean and Western diets. IMPORTANCE The human gastrointestinal microbiota functions as an important mediator of diet for host metabolism. To evaluate how consumed diets influence the gut environment, we carried out simultaneous interrogations of distal gut microbiota and metabolites in samples from healthy children in Egypt and the United States. While Egyptian children consumed a Mediterranean diet rich in plant foods, U.S. children consumed a Western diet high in animal protein, fats, and highly processed carbohydrates. Consistent with the consumed diets, Egyptian gut samples were enriched in polysaccharide-degrading microbes and end products of polysaccharide fermentation and U.S. gut samples were enriched in proteolytic microbes and end products of protein and fat metabolism. Thus, the intestinal microbiota might be selected on the basis of the diets that we consume, which can open opportunities to affect gut health through modulation of gut microbiota with dietary supplementations.

The effect of enhanced acetate influx on Synechocystis sp. PCC 6803 metabolism

BackgroundAcetate is a common microbial fermentative end-product, which can potentially be used as a supplementary carbon source to enhance the output of biotechnological production systems. This study focuses on the acetate metabolism of the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 which is unable to grow on acetate as a sole carbon source but still can assimilate it via acetyl-CoA—derived metabolic intermediates. In order to gain insight into the acetate uptake, associated limitations and metabolic effects, a heterologous acetate transporter ActP from Escherichia coli was introduced into Synechocystis to facilitate the transport of supplemented acetate from the medium into the cell.ResultsThe results show that enhanced acetate intake can efficiently promote the growth of the cyanobacterial host. The effect is apparent specifically under low-light conditions when the photosynthetic activity is low, and expected to result from increased availability of acetyl-CoA precursors, accompanied by changes induced in cellular glycogen metabolism which may include allocation of resources towards enhanced growth instead of glycogen accumulation. Despite the stimulated growth of the mutant, acetate is shown to suppress the activity of the photosynthetic apparatus, further emphasizing the contribution of glycolytic metabolism in the acetate-induced effect.ConclusionsThe use of acetate by the cyanobacterium Synechocystis sp. PCC 6803 is at least partially restricted by the import into the cell. This can be improved by the introduction of a heterologous acetate transporter into the system, thereby providing a potential advantage by expanding the scope of acetate utilization for various biosynthetic processes.

Cooking Has Variable Effects on the Fermentability in the Large Intestine of the Fraction of Meats, Grain Legumes, and Insects That Is Resistant to Digestion in the Small Intestine in an in Vitro Model of the Pig's Gastrointestinal Tract.

This study aimed to evaluate the fermentation in the large intestine of indigestible dietary protein sources from animal, insect, and plant origin using an in vitro model of the pig's gastrointestinal tract. Protein sources were used raw and after a cooking treatment. Results showed that the category of the ingredient (meats, insects, or grain legumes) exerts a stronger impact on enzymatic digestibility, fermentation patterns, and bacterial metabolites such as short-chain fatty acids (SCFA) and hydrogen sulfide (H2S) than the cooking treatment. The digestibility and the fermentation characteristics of insects were more affected by the cooking procedure than the other categories. Per gram of consumed food, ingredients from animal origin, namely, meats and insects, were associated with fewer fermentation end-products (gas, H2S, SCFA) than ingredients from plant origin, which is related to their higher small intestinal digestibility.

Wax Ester Fermentation and Its Application for Biofuel Production.

In Euglena cells under anaerobic conditions, paramylon, the storage polysaccharide, is promptly degraded and converted to wax esters. The wax esters synthesized are composed of saturated fatty acids and alcohols with chain lengths of 10-18, and the major constituents are myristic acid and myristyl alcohol. Since the anaerobic cells gain ATP through the conversion of paramylon to wax esters, the phenomenon is named "wax ester fermentation". The wax ester fermentation is quite unique in that the end products, i.e. wax esters, have relatively high molecular weights, are insoluble in water, and accumulate in the cells, in contrast to the common fermentation end products such as lactic acid and ethanol.A unique metabolic pathway involved in the wax ester fermentation is the mitochondrial fatty acid synthetic system. In this system, fatty acid are synthesized by the reversal of β-oxidation with an exception that trans-2-enoyl-CoA reductase functions instead of acyl-CoA dehydrogenase. Therefore, acetyl-CoA is directly used as a C2 donor in this fatty acid synthesis, and the conversion of acetyl-CoA to malonyl-CoA, which requires ATP, is not necessary. Consequently, the mitochondrial fatty acid synthetic system makes possible the net gain of ATP through the synthesis of wax esters from paramylon. In addition, acetyl-CoA is provided in the anaerobic cells from pyruvate by the action of a unique enzyme, oxygen sensitive pyruvate:NADP+ oxidoreductase, instead of the common pyruvate dehydrogenase multienzyme complex.Wax esters produced by anaerobic Euglena are promising biofuels because myristic acid (C14:0) in contrast to other algal produced fatty acids, such as palmitic acid (C16:0) and stearic acid (C18:0), has a low freezing point making it suitable as a drop-in jet fuel. To improve wax ester production, the molecular mechanisms by which wax ester fermentation is regulated in response to aerobic and anaerobic conditions have been gradually elucidated by identifying individual genes related to the wax ester fermentation metabolic pathway and by comprehensive gene/protein expression analysis. In addition, expression of the cyanobacterial Calvin cycle fructose-1,6-bisphosphatase/sedohepturose-1,7-bisphosphatase, in Euglena provided photosynthesis resulting in increased paramylon accumulation enhancing wax ester production. This chapter will discuss the biochemistry of the wax ester fermentation, recent advances in our understanding of the regulation of the wax ester fermentation and genetic engineering approaches to increase production of wax esters for biofuels.

Raw meat based diet influences faecal microbiome and end products of fermentation in healthy dogs.

BackgroundDietary intervention studies are required to deeper understand the variability of gut microbial ecosystem in healthy dogs under different feeding conditions and to improve diet formulations. The aim of the study was to investigate in dogs the influence of a raw based diet supplemented with vegetable foods on faecal microbiome in comparison with extruded food.MethodsEight healthy adult Boxer dogs were recruited and randomly divided in two experimental blocks of 4 individuals. Dogs were regularly fed a commercial extruded diet (RD) and starting from the beginning of the trial, one group received the raw based diet (MD) and the other group continued to be fed with the RD diet (CD) for a fortnight. After 14 days, the two groups were inverted, the CD group shifted to the MD and the MD shifted to the CD, for the next 14 days. Faeces were collected at the beginning of the study (T0), after 14 days (T14) before the change of diet and at the end of experimental period (T28) for DNA extraction and analysis of metagenome by sequencing 16SrRNA V3 and V4 regions, short chain fatty acids (SCFA), lactate and faecal score.ResultsA decreased proportion of Lactobacillus, Paralactobacillus (P < 0.01) and Prevotella (P < 0.05) genera was observed in the MD group while Shannon biodiversity Index significantly increased (3.31 ± 0.15) in comparison to the RD group (2.92 ± 0.31; P < 0.05). The MD diet significantly (P < 0.05) decreased the Faecal Score and increased the lactic acid concentration in the feces in comparison to the RD treatment (P < 0.01). Faecal acetate was negatively correlated with Escherichia/Shigella and Megamonas (P < 0.01), whilst butyrate was positively correlated with Blautia and Peptococcus (P < 0.05). Positive correlations were found between lactate and Megamonas (P < 0.05), Escherichia/Shigella (P < 0.01) and Lactococcus (P < 0.01).ConclusionThese results suggest that the diet composition modifies faecal microbial composition and end products of fermentation. The administration of MD diet promoted a more balanced growth of bacterial communities and a positive change in the readouts of healthy gut functions in comparison to RD diet.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-017-0981-z) contains supplementary material, which is available to authorized users.

Anaerobic biosynthesis of intermediates of reductive branch of tricarboxylic acids cycle by Escherichia coli strains with inactivated frdAB and sdhAB genes

The genes frdAB and sdhAB, which encode components of fumarate reductase and succinate dehydrogenase, have been deleted in a recombinant E. coli strain with the inactivated pathways of mixed-acid fermentation and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. Under anaerobic conditions, the parental strain efficiently converted glucose to succinic acid without synthesizing notable amounts of fumaric or malic acid. Upon individual deletion of the frdAB genes, the mutant strain fermented glucose to succinic acid less efficiently secreting notable amounts of malic and fumaric acids. Individual deletion of the sdhAB genes in the parental strain did not significantly affect the formation of the main fermentation end-product. The combined inactivation of fumarate reductase and succinate dehydrogenase in the constructed strain enhanced the anaerobic conversion of glucose to fumaric and malic acids with the activation of the glyoxylate bypass and decrease in the contribution of the reductive branch of the TCA cycle to the formation of the target products.

Volatile fatty acids and methane production from browse species of Algerian arid and semi-arid areas

ABSTRACTThe objective of the study was to determine the in vitro rumen fermentation end-products of 10 browse species. Serum bottles containing 500 mg of substrate, 10 ml of sheep rumen fluid and 40 ml of buffered medium were incubated for 24 h. After incubation, pH, methane and volatile fatty acid (VFA) productions were recorded. There were differences among feedstuffs (p < .05) in pH and VFA production. Astragalus gombo resulted in the highest and Stipa tenacissima in the lowest VFA production. Gas production was highest for Medicago sativa and lowest for S. tenacissima. Methane production (ml/g DM incubated) varied greatly. The lowest methane production was for S. tenacissima and Arthrocnemum macrostachyum (11.4 and 11.5 ml/g DM, respectively) and the highest for M. sativa and A. gombo (25.8 and 22.7 ml/g DM, respectively). The differences among species shrank when methane was expressed per ml of total gas produced or per mol of VFA produced. This indicates that a lower methane production would be due to a low fermentability of the substrate incubated, rather than to a specific inhibitory effect on methanogenesis. Hence, the 10 browse species studied herein would show little potential for mitigating ruminal methane production.

The gut-kidney axis.

The host-gut microbiota interaction has been the focus of increasing interest in recent years. It has been determined that this complex interaction is not only essential to many aspects of normal "mammalian" physiology but that it may also contribute to a multitude of ailments, from the obvious case of inflammatory bowel disease to (complex) diseases residing in organs outside the gut. An increasing body of evidence indicates that crosstalk between host and microbiota is pathophysiologically relevant in patients with chronic kidney disease (CKD). Interactions are bidirectional; on the one hand, uremia affects both the composition and metabolism of the gut microbiota and, on the other hand, important uremic toxins originate from microbial metabolism. In addition, gut dysbiosis may induce a disruption of the epithelial barrier, ultimately resulting in increased exposure of the host to endotoxins. Due to dietary restrictions and gastrointestinal dysfunctions, microbial metabolism shifts to a predominantly proteolytic fermentation pattern in CKD. Indoxyl sulfate and p-cresyl sulfate, both end-products of protein fermentation, and trimethylamine-N-oxide, an end-product of microbial choline and carnitine metabolism, are prototypes of uremic toxins originating from microbial metabolism. The vascular and renal toxicity of these co-metabolites has been demonstrated extensively in experimental and clinical studies. These co-metabolites are an appealing target for adjuvant therapy in CKD. Treatment options include dietary therapy, prebiotics, probiotics and host and bacterial enzyme inhibitors. Final proof of the concept should come from randomized controlled and adequately powered intervention studies.