Whole-Grain Rye and Wheat Affect Some Markers of Gut Health without Altering the Fecal Microbiota in Healthy Overweight Adults: A 6-Week Randomized Trial.

Background: Whole grains have shown potential for improving gut health, but evidence comparing different whole-grain types is lacking.Objective: We investigated whether whole-grain wheat (WGW) and whole-grain rye (WGR) improve gut health in different ways compared to refined wheat (RW), with the primary outcomes of microbiota composition and gastrointestinal (GI) symptoms.Methods: In a randomized parallel trial, 70 healthy adults (in means ± SDs; aged 51.0 ± 9.4 y, body mass index [BMI (in kg/m2)] 27.8 ± 1.9, 32:38 men:women) replaced cereal foods from their habitual diet with WGR, WGW, or RW (control). Before and after a 6-wk intervention, a spot stool sample was collected and analyzed for short-chain fatty acids and microbiota composition through the use of 16S ribosomal RNA gene-targeted high-throughput amplicon sequencing. GI symptoms and stool regularity were evaluated by questionnaires at baseline and after weeks 2, 4, and 6.Results: Intakes of whole grains were 145.2 ± 75.9, 124.2 ± 57.3, and 5.4 ± 3.2 g/d in the WGW, WGR, and RW groups, respectively. Gut microbiota composition was not affected by diet. The relative change in fecal butyrate decreased in the RW (-38%) group compared to the WGW (25%, P = 0.014) and WGR groups (-1%, P = 0.037). Other short-chain fatty acids were unaffected. Flatulence was more frequent following intake of WGW (OR: 2.06, 95% CI: 1.03, 4.17) and WGR (OR: 2.62, 95% CI: 1.35, 5.22) compared to RW, whereas bloating was less frequent following WGW (OR: 0.38, 95% CI: 0.18, 0.80) and WGR (OR: 0.34, 95% CI: 0.16, 0.72). Stool frequency increased following WGR but not WGW, compared to RW in weeks 2 (0.4 defecations/d, P = 0.049) and 4 (0.5 defecations/d, P = 0.043), but not in week 6. The WGW and WGR groups did not differ from each other in any of the variables tested.Conclusion: Regular consumption of WGR and WGW affected fecal butyrate concentration and gastrointestinal symptoms in healthy overweight adults, supporting the hypothesis that WGR and WGW can be included in the diet equally to maintain gut health. This trial was registered at clinicaltrials.gov as NCT02358122.

Microbial Production of Short Chain Fatty Acids from Lignocellulosic Biomass: Current Processes and Market.

Biological production of organic acids from conversion of biomass derivatives has received increased attention among scientists and engineers and in business because of the attractive properties such as renewability, sustainability, degradability, and versatility. The aim of the present review is to summarize recent research and development of short chain fatty acids production by anaerobic fermentation of nonfood biomass and to evaluate the status and outlook for a sustainable industrial production of such biochemicals. Volatile fatty acids (VFAs) such as acetic acid, propionic acid, and butyric acid have many industrial applications and are currently of global economic interest. The focus is mainly on the utilization of pretreated lignocellulosic plant biomass as substrate (the carbohydrate route) and development of the bacteria and processes that lead to a high and economically feasible production of VFA. The current and developing market for VFA is analyzed focusing on production, prices, and forecasts along with a presentation of the biotechnology companies operating in the market for sustainable biochemicals. Finally, perspectives on taking sustainable product of biochemicals from promise to market introduction are reviewed.

Effect of in situ acids removal on mixed glucose and xylose fermentation by Clostridium tyrobutyricum.

In the present study, the effect of potassium ions and increasing concentrations of glucose and xylose on the growth of a strain of Clostridium tyrobutyricum, adapted to wheat straw hydrolysate, was investigated. Application of continuous fermentation of a mixture of glucose and xylose and in situ acid removal by reverse electro enhanced dialysis (REED) was investigated as a method to alleviate potassium and end-product inhibition and consequently enhance the sugar consumption rates and butyric acid productivity. It was found that glucose and xylose were not inhibitory up to a concentration of 50 and 37 g L(-1) respectively, and that they were consumed at comparable rates when fermented alone. However, continuous fermentation of a mixture of glucose and xylose resulted in a significantly decreased xylose consumption rate compared to that of glucose alone, supporting the conclusion that C. tyrobutyricum has a lower affinity for xylose than for glucose. Potassium ions negatively affected the effective maximum growth rate of C. tyrobutyricum at concentrations higher than 5 g L(-1) exhibiting a non-competitive type of inhibition. Continuous fermentation of a glucose and xylose mixture with simultaneous acid removal by REED resulted in a two to threefold increase of the glucose consumption rate, while the xylose consumption rate was enhanced sixfold compared to continuous fermentation without in situ acid removal. Similarly, butyric acid productivity was enhanced by a factor of 2-3, while the yield remained unaffected.

Production of 1,3-PDO and butanol by a mutant strain of Clostridium pasteurianum with increased tolerance towards crude glycerol.

The production of biodiesel results in a concomitant production of crude glycerol (10% w/w). Clostridium pasteurianum can utilize glycerol as sole carbon source and converts it into 1,3-propanediol, ethanol, butanol, and CO2. Reduced growth and productivities on crude glycerol as compared to technical grade glycerol have previously been observed. In this study, we applied random mutagenesis mediated by ethane methyl sulfonate (EMS) to develop a mutant strain of C. pasteurianum tolerating high concentrations of crude glycerol. At an initial crude glycerol concentration of 25 g/l the amount of dry cell mass produced by the mutant strain was six times higher than the amount produced by the wild type. Growth of the mutant strain was even detected at an initial crude glycerol concentration of 105 g/l. A pH controlled reactor with in situ removal of butanol by gas-stripping was used to evaluate the performance of the mutant strain. Utilizing stored crude glycerol, the mutant strain showed significantly increased rates compared to the wild type. A maximum glycerol utilization rate of 7.59 g/l/h was observed along with productivities of 1.80 g/l/h and 1.21 g/l/h of butanol and 1,3-PDO, respectively. These rates are higher than what previously has been published for C. pasteurianum growing on technical grade glycerol in fed batch reactors. In addition, high yields of the main products (butanol and 1,3-PDO) were detected and these two products were efficiently separated in two steams using gas-stripping.

Fermentation of crude glycerol from biodiesel production by Clostridium pasteurianum.

Clostridium pasteurianum can utilize glycerol as the sole carbon source for the production of butanol and 1,3-propanediol. Crude glycerol derived from biodiesel production has been shown to be toxic to the organism even in low concentrations. By examination of different pretreatments we found that storage combined with activated stone carbon addition facilitated the utilization of crude glycerol. A pH-controlled reactor with in situ removal of butanol by gas stripping was used to evaluate the performance. The fermentation pattern on pretreated crude glycerol was quite similar to that on technical grade glycerol. C. pasteurianum was able to utilize 111 g/l crude glycerol. The average consumption rate was 2.49 g/l/h and maximum consumption rate was 4.08 g/l/h. At the maximal glycerol consumption rate butanol was produced at 1.3 g/l/h. These rates are higher than those previously reported for fermentations on technical grade glycerol by the same strain. A process including pretreatment and subsequent fermentation of the crude glycerol could be usable for industrial production of butanol by C. pasteurianum.

In vitro fermentation of arabinoxylan-derived carbohydrates by bifidobacteria and mixed fecal microbiota.

Bifidobacterium adolescentis ATCC 15703, Bifidobacterium breve ATCC 15700, Bifidobacterium longum ATCC 15707, and human fecal microbiota were cultivated in vitro with d-xylose, l-arabinose, xylo-oligosaccharides (XOS), and arabinoxylo-oligosaccharides (AXOS) as carbon sources. The pH, formation of volatile fatty acids, and carbohydrate utilization profiles were followed. In the pure bifidobacteria cultures optical density and in the fecal slurries pressure and H(2) were also detected. A differing substrate preference was observed among the various bifidobacteria strains. B. adolescentis grew on XOS, slowly on d-xylose, but not on l-arabinose. In contrast, B. longum preferred l-arabinose and did not grow on pure d-xylose or XOS. Both strains were able to utilize AXOS but with differing strategies, since after the cleavage of l-arabinose B. adolescentis consumed the XOS formed, whereas B. longum fermented the l-arabinose released. B. breve grew poorly on all of the substrates provided. A bifidobacterial mixture and the fecal microbiota were able to utilize pure singly substituted AXOS almost completely, but pure AXOS with a doubly substituted xylose residue was fermented only by the fecal microbiota. Thus, AXOS appear to be potential candidates for slowly fermenting prebiotics, but their prebiotic effects may be dependent on the type of arabinose substitution and the presence of other carbohydrates.

Coproduction of bioethanol with other biofuels.

Large scale transformation of biomass to more versatile energy carriers has most commonly been focused on one product such as ethanol or methane. Due to the nature of the biomass and thermodynamic and biological constraints, this approach is not optimal if the energy content of the biomass is supposed to be exploited maximally. In natural ecosystems, biomass is degraded to numerous intermediary compounds, and we suggest that this principle is utilized in biorefinery concepts, which could provide different fuels with different end use possibilities. In this chapter we describe one of the first pilot-scale biorefineries for multiple fuel production and also discuss perspectives for further enhancement of biofuel yields from biomass. The major fuels produced in this refinery are ethanol, hydrogen, and methane. We also discuss the applicability of our biorefinery concept as a bolt-on plant on conventional corn- or grain-based bioethanol plants, and suggest that petroleum-base refineries and biorefineries appropriately can be coupled during the transition period from a fossil fuel to a renewable fuel economy.

Archaeal diversity in Icelandic hot springs.

Whole-cell density gradient extractions from three solfataras (pH 2.5) ranging in temperature from 81 to 90 degrees C and one neutral hot spring (81 degrees C, pH 7) from the thermal active area of Hveragerethi (Iceland) were analysed for genetic diversity and local geographical variation of Archaea by analysis of amplified 16S rRNA genes. In addition to the three solfataras and the neutral hot spring, 10 soil samples in transects of the soil adjacent to the solfataras were analysed using terminal restriction fragment length polymorphism (t-RFLP). The sequence data from the clone libraries in combination with 14 t-RFLP profiles revealed a high abundance of clones clustering together with sequences from the nonthermophilic I.1b group of Crenarchaeota. The archaeal diversity in one solfatara was high; 26 different RFLP patterns were found using double digestion of the PCR products with restriction enzymes AluI and BsuRI. The sequenced clones from this solfatara belonged to Sulfolobales, Thermoproteales or were most closest related to sequences from uncultured Archaea. Sequences related to group I.1b were not found in the neutral hot spring or the hyperthermophilic solfatara (90 degrees C).

Specific single-cell isolation and genomic amplification of uncultured microorganisms.

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.

Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network.

The role of dynamin and so-called accessory proteins in endocytosis is well established. However, molecular details of the function(s) of dynamin II at the Golgi are largely unclear. We demonstrate that the ubiquitously expressed syndapin II isoform interacts with the proline-rich domain (PRD) of dynamin II through its Src-homology 3 (SH3) domain. Co-immunoprecipitation of endogenous syndapin II and dynamin II, and successful reconstitutions of such complexes at membranes in COS-7 cells, show the in vivo relevance of the interaction. Syndapin II can associate with Golgi membranes and this association increases upon Golgi exit block. Brefeldin A treatment clearly shows that the observed perinuclear localization of syndapin II co-localizing with syntaxin 6 reflects the Golgi complex and that it requires functional integrity of the Golgi. Syndapins are crucial for Golgi vesicle formation because anti-syndapin antibodies, used either in in vitro reconstitutions or in living cells, inhibited this process. Both types of assays additionally revealed the essential role of syndapin II SH3 interactions with the dynamin II PRD in vesicle formation. An excess of the syndapin SH3 domain strongly inhibited budding from Golgi membranes in vitro. Likewise, overexpression of the syndapin SH3 domain or of a dynamin II variant incapable of associating with syndapin II (dynamin IIDeltaPRD) impaired trafficking of vesicular stomatitis virus glycoprotein (VSVG)-GFP in vivo. By contrast, full-length syndapin II-l had no negative effect, and instead promoted VSVG-GFP export from the Golgi. Importantly, a cytosolic fraction containing endogenous syndapin-dynamin complexes was sufficient to promote vesicle formation from Golgi membranes in a syndapin-dependent manner. Thus, syndapin-dynamin complexes are crucial and sufficient to promote vesicle formation from the trans-Golgi network.

An improved method for single cell isolation of prokaryotes from meso-, thermo- and hyperthermophilic environments using micromanipulation.

This study presents an improved system that enables isolation of single viable prokaryotic cells from a mixture of cells. The system is based on an inverted microscope, a microinjector and a micromanipulator. The isolated cell is captured in a microcapillary from a volume of 400 mul and transferred to an appropriate growth medium. Validation of the system was performed using two fluorescent strains: Pseudomonas putida expressing red fluorescent protein (DsRed), and Escherichia coli expressing green fluorescent protein (GFP). A mixture (100:1) of the constructed fluorescent strains was subjected to isolation experiments and nine out of ten individually isolated cells yielded axenic cultures of E. coli. Upon construction and validation, the system was used to isolate and subsequently cultivate axenic cultures of the thermophilic Archaeon Metallosphaera sedula and the hyperthermophilic Archaeon Sulfolobus solfataricus from enriched hot spring samples. The high efficiency of single-cell isolation and cultivation demonstrated over a range of temperatures-90% (30 degrees C), 85% (70 degrees C) and 95% (80 degrees C)-from different environments is probably due to the elimination of osmotic stress and limitation of temperature fluctuations during the isolation process, as a result of the large sample volume from which the cells are isolated.

Diversity of thermophilic and non-thermophilic Crenarchaeota at 80 degrees C.

A hot spring in the solfataric field of Pisciarelli (Naples-Italy) was analysed for Archaeal diversity. Total DNA was extracted from the environment, archaeal 16S rRNA genes were amplified with Archaea specific primers, and a clone library consisting of 201 clones was established. The clones were grouped in 10 different groups each representing a specific band pattern using restriction fragment length polymorphism (RFLP). Members of all 10 groups were sequenced and phylogenetically analyzed. Surprisingly, a high abundance of clones belonging to non-thermophilic Crenarchaeal clusters were detected together with the thermophilic archaeon Acidianus infernus in this thermophilic environment. Neither Sulfolobus species nor other hyperthermophilic Crenarchaeota were detected in the clone library. The relative abundance of the sequenced clones was confirmed by terminal restriction fragment analyses. Amplification of 16S rRNA genes from Archaea transferred from the surrounding environment was considered negligible because DNA from non-thermophilic Crenarchaeota incubated under conditions similar to the solfatara could not be PCR amplified after 5 min.

Archaea in protozoa and metazoa.

The presence of Archaea is currently being explored in various environments, including extreme geographic positions and eukaryotic habitats. Methanogens are the dominating archaeal organisms found in most animals, from unicellular protozoa to humans. Many methanogens can contribute to the removal of hydrogen, thereby improving the efficiency of fermentation or the reductive capacity of energy-yielding reactions. They may also be involved in tissue damage in periodontal patients. Recent molecular studies demonstrated the presence of Archaea other than methanogens in some animals-but so far, not in humans. The roles of these microorganisms have not yet been established. In the present review, we present the state of the art regarding the archaeal microflora in animals.

Mesophilic and thermophilic anaerobic digestion of primary and secondary sludge. Effect of pre-treatment at elevated temperature.

Anaerobic digestion is an appropriate technique for the treatment of sludge before final disposal and it is employed worldwide as the oldest and most important process for sludge stabilization. In general, mesophilic anaerobic digestion of sewage sludge is more widely used compared to thermophilic digestion. Furthermore, thermal pre-treatment is suitable for the improvement of stabilization, enhancement of dewatering of the sludge, reduction of the numbers of pathogens and could be realized at relatively low cost especially at low temperatures. The present study investigates (a) the differences between mesophilic and thermophilic anaerobic digestion of sludge and (b) the effect of the pre-treatment at 70 degrees C on mesophilic and thermophilic anaerobic digestion of primary and secondary sludge. The pre-treatment step showed very positive effect on the methane potential and production rate upon subsequent thermophilic digestion of primary sludge. The methane production rate was mostly influenced by the pre-treatment of secondary sludge followed by mesophilic and thermophilic digestion whereas the methane potential only was positively influenced when mesophilic digestion followed. Our results suggest that the selection of the pre-treatment duration as well as the temperature of the subsequent anaerobic step for sludge stabilization should depend on the ratio of primary to secondary sludge.