The effects of sustained fitness improvement on the gut microbiome: A longitudinal, repeated measures case-study approach.

The athlete gut microbiome differs from that of non-athletes in its composition and metabolic function. Short-term fitness improvement in sedentary adults does not replicate the microbiome characteristics of athletes. The objective of this study was to investigate whether sustained fitness improvement leads to pronounced alterations in the gut microbiome. This was achieved using a repeated-measures, case-study approach that examined the gut microbiome of two initially unfit volunteers undertaking progressive exercise training over a 6-month period. Samples were collected every two weeks, and microbiome, metabolome, diet, body composition, and cardiorespiratory fitness data were recorded. Training culminated in both participants completing their respective goals (a marathon or Olympic-distance triathlon) with improved body composition and fitness parameters. Increases in gut microbiota α-diversity occurred with sustained training and fluctuations occurred in response to training events (eg, injury, illness, and training peaks). Participants' BMI reduced during the study and was significantly associated with increased urinary measurements of N-methyl nicotinate and hippurate, and decreased phenylacetylglutamine. These results suggest that sustained fitness improvements support alterations to gut microbiota and physiologically-relevant metabolites. This study provides longitudinal analysis of the gut microbiome response to real-world events during progressive fitness training, including intercurrent illness and injury.

Four men in a boat: Ultra-endurance exercise alters the gut microbiome.

OBJECTIVES: Compositional and functional adaptions occur in the gut microbiome in response to habitual physical activity. The response of the gut microbiome to sustained, intense exercise in previously active individuals, however, is unknown. This study aimed to prospectively explore the gut microbiome response of four well-trained male athletes to prolonged, high intensity trans-oceanic rowing, describing changes in microbial diversity, abundance and metabolic capacity. DESIGN: A prospective, repeated-measures, within-subject report. METHODS: Serial stool samples were obtained from four male athletes for metagenomic whole-genome shotgun sequencing to record microbial community structure and relevant functional gene profiles before, during and after a continuous, unsupported 33-day, 5000 km transoceanic rowing race. Calorific intake and macronutrient composition were recorded by validated food frequency questionnaire and anthropometry was determined by body composition analysis and cardiorespiratory testing. RESULTS: Microbial diversity increased throughout the ultra-endurance event. Variations in taxonomic composition included increased abundance of butyrate producing species and species associated with improved metabolic health, including improved insulin sensitivity. The functional potential of bacterial species involved in specific amino and fatty acid biosynthesis also increased. Many of the adaptions in microbial community structure and metaproteomics persisted at three months follow up. CONCLUSIONS: These findings demonstrate that prolonged, intense exercise positively influences gut microbial diversity, increases the relative abundance of some bacterial species and up-regulates the metabolic potential of specific pathways expressing microbial gene products. These adaptions may play a compensatory role in controlling the physiological stress associated with sustained exertion as well as negating the deleterious consequences accompanying endurance exercise.

A Repeated-measures Case Series of Physiological Responses to a Transoceanic Rowing Race.

This repeated-measures case series describes the changes in cardiorespiratory fitness, body composition and systemic inflammation in 4 well-trained athletes pre- and post-completion of an unsupported transatlantic rowing race. The acute effects of endurance exercise have been well described previously, but the enduring consequences of ultra-endurance on the cardiorespiratory, metabolic and immune systems are largely unknown. This study explores these physiological adaptations following 2 weeks of recovery. Cardiorespiratory fitness testing, body composition analysis, and blood sampling for inflammatory cytokines were recorded immediately before race departure and repeated 14 days following race completion. Mean VO2max (ml/kg/min) was similar pre- (48.2±2.8) and post-race (46.7±1.5). Heart rate responses were equivalent at incremental workloads. Mean blood lactate (mmol/L) was higher at low to moderate power outputs and lower at maximal effort (14.6±1.85 vs. 13.1±2.5). Percentage body fat (17.7 ± 7.9 vs. 16.2±7.4) was analogous to pre-race analysis. Low-grade inflammation persisted, indicated by an increase in IL-1ß (69%), IL-8 (10%), TNF-α (8%), IL-6 (5.4%), and C-reactive protein (22.4%). VO2max and heart rate responses were similar pre- and post-race, but sub-maximal efficiency measures of cardiorespiratory fitness were consistent with persistent fatigue. Body composition had returned to baseline but low-grade systemic inflammation persisted. Persistent pro-inflammatory cytokinaemia is known to exert deleterious consequences on immune, metabolic, and psychological function. Adequate recovery is necessary to re-establish inflammatory homeostasis, and the results of this study may inform these decisions.

A Prospective Metagenomic and Metabolomic Analysis of the Impact of Exercise and/or Whey Protein Supplementation on the Gut Microbiome of Sedentary Adults.

Many components of modern living exert influence on the resident intestinal microbiota of humans with resultant impact on host health. For example, exercise-associated changes in the diversity, composition, and functional profiles of microbial populations in the gut have been described in cross-sectional studies of habitual athletes. However, this relationship is also affected by changes in diet, such as changes in dietary and supplementary protein consumption, that coincide with exercise. To determine whether increasing physical activity and/or increased protein intake modulates gut microbial composition and function, we prospectively challenged healthy but sedentary adults with a short-term exercise regime, with and without concurrent daily whey protein consumption. Metagenomics- and metabolomics-based assessments demonstrated modest changes in gut microbial composition and function following increases in physical activity. Significant changes in the diversity of the gut virome were evident in participants receiving daily whey protein supplementation. Results indicate that improved body composition with exercise is not dependent on major changes in the diversity of microbial populations in the gut. The diverse microbial characteristics previously observed in long-term habitual athletes may be a later response to exercise and fitness improvement. IMPORTANCE The gut microbiota of humans is a critical component of functional development and subsequent health. It is important to understand the lifestyle and dietary factors that affect the gut microbiome and what impact these factors may have. Animal studies suggest that exercise can directly affect the gut microbiota, and elite athletes demonstrate unique beneficial and diverse gut microbiome characteristics. These characteristics are associated with levels of protein consumption and levels of physical activity. The results of this study show that increasing the fitness levels of physically inactive humans leads to modest but detectable changes in gut microbiota characteristics. For the first time, we show that regular whey protein intake leads to significant alterations to the composition of the gut virome.

Modelling and understanding powder flow properties and compactability of selected active pharmaceutical ingredients, excipients and physical mixtures from critical material properties.

The development of solid dosage forms and manufacturing processes are governed by complex physical properties of the powder and the type of pharmaceutical unit operation the manufacturing processes employs. Suitable powder flow properties and compactability are crucial bulk level properties for tablet manufacturing by direct compression. It is also generally agreed that small scale powder flow measurements can be useful to predict large scale production failure. In this study, predictive multilinear regression models were effectively developed from critical material properties to estimate static powder flow parameters from particle size distribution data for a single component and for binary systems. A multilinear regression model, which was successfully developed for ibuprofen, also efficiently predicted the powder flow properties for a range of batches of two other active pharmaceutical ingredients processed by the same manufacturing route. The particle size distribution also affected the compactability of ibuprofen, and the scope of this work will be extended to the development of predictive multivariate models for compactability, in a similar manner to the approach successfully applied to flow properties.

Use of a mannitol rich ensiled grass press juice (EGPJ) as a sole carbon source for polyhydroxyalkanoates (PHAs) production through high cell density cultivation.

This study demonstrates the use of a mannitol rich ensiled grass press juice (EGPJ) as a renewable carbon substrate for polyhydroxyalkanoates (PHA) production in shaking flask experiments and fed-batch stirred tank reactor cultivations. Fed-batch cultivations of Burkholderia sacchari IPT101 using EGPJ as sole carbon source produced 44.5 g/L CDW containing 33% polyhydroxybutyrate (PHB) in 36 h, while Pseudomonas chlororaphis IMD555 produced a CDW of 37 g/L containing 10% of medium chain length polyhydroxyalkanoates (mcl-PHA) in 34 h. PHB and mcl-PHA extracted from B. sacchari IPT101 and P. chlororaphis IMD555, grown on EGPJ, had a molecular weight of 548 kg/mol and 115.4 kg/mol, respectively. While mcl-PHA can be produced from EGPJ, PHB production is more interesting as there is a 4-fold higher volumetric productivity compared to mcl-PHA.

Fed-batch strategies using butyrate for high cell density cultivation of Pseudomonas putida and its use as a biocatalyst.

A mathematically based fed-batch bioprocess demonstrated the suitability of using a relatively cheap and renewable substrate (butyric acid) for Pseudomonas putida CA-3 high cell density cultivation. Butyric acid fine-tuned addition is critical to extend the fermentation run and avoid oxygen consumption while maximising the biomass volumetric productivity. A conservative submaximal growth rate (µ of 0.25 h(-1)) achieved 71.3 g L(-1) of biomass after 42 h of fed-batch growth. When a more ambitious feed rate was supplied in order to match a µ of 0.35 h(-1), the volumetric productivity was increased to 2.0 g L(-1) h(-1), corresponding to a run of 25 h and 50 g L(-1) of biomass. Both results represent the highest biomass and the best biomass volumetric productivity with butyrate as a sole carbon source. However, medium chain length polyhydroxyalkanoate (mcl-PHA) accumulation with butyrate grown cells is low (4 %). To achieve a higher mcl-PHA volumetric productivity, decanoate was supplied to butyrate grown cells. This strategy resulted in a PHA volumetric productivity of 4.57 g L(-1) h(-1) in the PHA production phase and 1.63 g L(-1) h(-1)over the lifetime of the fermentation, with a maximum mcl-PHA accumulation of 65 % of the cell dry weight.

Identification and characterization of an acyl-CoA dehydrogenase from Pseudomonas putida KT2440 that shows preference towards medium to long chain length fatty acids.

Diverse and elaborate pathways for nutrient utilization, as well as mechanisms to combat unfavourable nutrient conditions make Pseudomonas putida KT2440 a versatile micro-organism able to occupy a range of ecological niches. The fatty acid degradation pathway of P. putida is complex and correlated with biopolymer medium chain length polyhydroxyalkanoate (mcl-PHA) biosynthesis. Little is known about the second step of fatty acid degradation (ß-oxidation) in this strain. In silico analysis of its genome sequence revealed 21 putative acyl-CoA dehydrogenases (ACADs), four of which were functionally characterized through mutagenesis studies. Four mutants with insertionally inactivated ACADs (PP_1893, PP_2039, PP_2048 and PP_2437) grew and accumulated mcl-PHA on a range of fatty acids as the sole source of carbon and energy. Their ability to grow and accumulate biopolymer was differentially negatively affected on various fatty acids, in comparison to the wild-type strain. Inactive PP_2437 exhibited a pattern of reduced growth and PHA accumulation when fatty acids with lengths of 10 to 14 carbon chains were used as substrates. Recombinant expression and biochemical characterization of the purified protein allowed functional annotation in P. putida KT2440 as an ACAD showing clear preference for dodecanoyl-CoA ester as a substrate and optimum activity at 30 °C and pH 6.5-7.

Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate.

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.

Conversion of grass biomass into fermentable sugars and its utilization for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas strains.

This study investigated the potential of grass biomass as a feedstock for mcl-PHA production. Pretreatments (2% NaOH at 120°C or hot water at 120°C) of perennial ryegrass were employed alone or in combination with sodium chlorite/acetic acid (SC/AA) delignification to evaluate the enzymatic digestibility and subsequent utilization of resultant sugars by Pseudomonas strains. NaOH pretreated sample had better digestibility than raw and hot water treated samples and this hydrolysate supported good growth of all tested strains with limited mcl-PHA (6-17% of cell dry mass (CDM)) accumulation. Digestibility of both untreated and pretreated samples was improved after SC/AA delignification and produced glucose (74-77%) rich hydrolysates. Tested strains accumulated 20-34% of CDM as PHA when these hydrolysates were used as sole carbon and energy source. CDM and PHA yields obtained for these strains when tested with laboratory grade sugars was similar to that achieved with grass derived sugars.

Preoperative evaluation of the maxillary sinus roof as a guide for posterior ethmoid and sphenoid sinus surgery.

BACKGROUND: The maxillary sinus roof has long been regarded as a reliable reference point for both endoscopic and skull base surgery. OBJECTIVE: This study was designed to evaluate the maxillary sinus roof as a preoperative guide for posterior ethmoid and sphenoid sinus surgery. METHOD: This was a cross-sectional observational study of 50 randomly selected computed tomographic (CT) sinus scans from patients previously seen at a tertiary rhinology clinic. Each side of the sinuses was treated independently, giving a total of 100 sides for analysis. Using the medial maxillary sinus roof as a fixed reference point, the vertical distance to the posterior ethmoid skull base, natural sphenoid ostium, and anterior sphenoid roof and floor were measured. In addition, the maxillary sinus to posterior ethmoid height ratio was calculated for each patient. RESULTS: Relative to the medial maxillary sinus roof, the mean vertical height of the posterior ethmoid skull base was 14.08 ± 3.03 mm. Using this same reference point, the mean vertical distance to the natural sphenoid ostium, sphenoid roof, and sphenoid floor was 2.76 ± 2.80 mm, 12.18 ± 3.20 mm, and 5.94 ± 2.94 mm, respectively. The mean maxillary sinus to posterior ethmoid height ratio was 2.49:1. CONCLUSION: The medial maxillary sinus roof is a reliable preoperative reference point for guiding safe surgical entry into the posterior ethmoid and sphenoid sinus. In addition to providing a margin of safety away from the skull base, it may also help with localization of the natural sphenoid ostium. A classification scheme for evaluating posterior ethmoid height is proposed.

Development of a bioprocess to convert PET derived terephthalic acid and biodiesel derived glycerol to medium chain length polyhydroxyalkanoate.

Sodium terephthalate (TA) produced from a PET pyrolysis product and waste glycerol (WG) from biodiesel manufacture were supplied to Pseudomonas putida GO16 in a fed-batch bioreactor. Six feeding strategies were employed by altering the sequence of TA and WG feeding. P. putida GO16 reached 8.70 g/l cell dry weight (CDW) and 2.61 g/l PHA in 48 h when grown on TA alone. When TA and WG were supplied in combination, biomass productivity (g/l/h) was increased between 1.3- and 1.7-fold and PHA productivity (g/l/h) was increased 1.8- to 2.2-fold compared to TA supplied alone. The monomer composition of the PHA accumulated from TA or WG was predominantly composed of 3-hydroxydecanoic acid. PHA monomers 3-hydroxytetradeeanoic acid and 3-hydroxytetradecenoic acid were not present in PHA accumulated from TA alone but were present when WG was supplied to the fermentation. When WG was either the sole carbon source or the predominant carbon source supplied to the fermentation the molecular weight of PHA accumulated was lower compared to PHA accumulated when TA was supplied as the sole substrate. Despite similarities in data for the properties of the polymers, PHAs produced with WG present in the PHA accumulation phase were tacky while PHA produced where TA was the sole carbon substrate in the polymer accumulation phase exhibited little or no tackiness at room temperature. The co-feeding of WG to fermentations allows for increased utilisation of TA. The order of feeding of WG and TA has an effect on TA utilisation and polymer properties.

Up-cycling of PET (polyethylene terephthalate) to the biodegradable plastic PHA (polyhydroxyalkanoate).

The conversion of the petrochemical polymer polyethylene terephthalate (PET) to a biodegradable plastic polyhydroxyal-kanoate (PHA) is described here. PET was pyrolised at 450 degrees C resulting in the production of a solid, liquid, and gaseous fraction. The liquid and gaseous fractions were burnt for energy recovery, whereas the solid fraction terephthalic acid (TA) was used as the feedstock for bacterial production of PHA. Strains previously reported to grow on TA were unable to accumulate PHA. We therefore isolated bacteria from soil exposed to PET granules at a PET bottle processing plant From the 32 strains isolated, three strains capable of accumulation of medium chain length PHA (mclPHA) from TA as a sole source of carbon and energy were selected for further study. These isolates were identified using 16S rDNA techniques as P. putida (GO16), P. putida (GO19), and P. frederiksbergensis (GO23). P. putida GO16 and GO19 accumulate PHA composed predominantly of a 3-hydroxydecanoic acid monomer while P. frederiksbergensis GO23 accumulates 3-hydroxydecanoic acid as the predominant monomer with increased amounts of 3-hydroxydodecanoic acid and 3-hydroxydodecenoic acid compared to the other two strains. PHA was detected in all three strains when nitrogen depleted below detectable levels in the growth medium. Strains GO16 and GO19 accumulate PHA at a maximal rate of approximately 8.4 mg PHA/l/h for 12 h before the rate of PHA accumulation decreased dramatically. Strain GO23 accumulates PHA at a lower maximal rate of 4.4 mg PHA/l/h but there was no slow down in the rate of PHA accumulation over time. Each of the PHA polymers is a thermoplastic with the onset of thermal degradation occurring around 308 degrees C with the complete degradation occurring by 370 degrees C. The molecular weight ranged from 74 to 123 kDa. X-ray diffraction indicated crystallinity of the order of 18-31%. Thermal analysis shows a low glass transition (-53 degrees C) with a broad melting endotherm between 0 and 45 degrees C.

The conversion of BTEX compounds by single and defined mixed cultures to medium-chain-length polyhydroxyalkanoate.

Here, we report the use of petrochemical aromatic hydrocarbons as a feedstock for the biotechnological conversion into valuable biodegradable plastic polymers--polyhydroxyalkanoates (PHAs). We assessed the ability of the known Pseudomonas putida species that are able to utilize benzene, toluene, ethylbenzene, p-xylene (BTEX) compounds as a sole carbon and energy source for their ability to produce PHA from the single substrates. P. putida F1 is able to accumulate medium-chain-length (mcl) PHA when supplied with toluene, benzene, or ethylbenzene. P. putida mt-2 accumulates mcl-PHA when supplied with toluene or p-xylene. The highest level of PHA accumulated by cultures in shake flask was 26% cell dry weight for P. putida mt-2 supplied with p-xylene. A synthetic mixture of benzene, toluene, ethylbenzene, p-xylene, and styrene (BTEXS) which mimics the aromatic fraction of mixed plastic pyrolysis oil was supplied to a defined mixed culture of P. putida F1, mt-2, and CA-3 in the shake flasks and fermentation experiments. PHA was accumulated to 24% and to 36% of the cell dry weight of the shake flask and fermentation grown cultures respectively. In addition a three-fold higher cell density was achieved with the mixed culture grown in the bioreactor compared to shake flask experiments. A run in the 5-l fermentor resulted in the utilization of 59.6 g (67.5 ml) of the BTEXS mixture and the production of 6 g of mcl-PHA. The monomer composition of PHA accumulated by the mixed culture was the same as that accumulated by single strains supplied with single substrates with 3-hydroxydecanoic acid occurring as the predominant monomer. The purified polymer was partially crystalline with an average molecular weight of 86.9 kDa. It has a thermal degradation temperature of 350 degrees C and a glass transition temperature of -48.5 degrees C.