Development of chemical-based reference standards for rooibos and honeybush aroma lexicons.

The honeybush sensory wheel was revised, using a large sample set (n = 585) comprising of the major commercial Cyclopia species, i.e. C. intermedia, C. subternata and C. genistoides. Only positive and negative aroma attributes were included in the wheel. Chemicals were identified to serve as reference standards for the honeybush (Cyclopia spp.) lexicon. Similarly, chemical-based reference standards were identified for the rooibos (Aspalathus linearis) lexicon. From a comprehensive literature search and gas chromatography-mass spectrometry analyses, chemicals were screened by an expert panel for their suitability in terms of typicality of the target aroma. Each chemical was evaluated in a 'base tea' and compared to a specific 'reference tea' exhibiting a high intensity of the target aroma. A total of 30 and 44 chemicals for rooibos and honeybush, respectively, were selected for validation by a trained panel. Descriptive sensory analysis was conducted to assign typicality and intensity scores for each chemical representing a target aroma attribute. Several chemicals were identified as suitable reference standards for the following aroma notes: isophorone ('rooibos-woody'), maltyl isobutyrate ('caramel'), cis-3-hexenol ('green grass'), 4-oxoisophorone ('seaweed') and 2,4,6-trichloroanisole ('musty/mouldy') for rooibos; and 2-acetyl-5-methylfuran ('woody'), levulinic acid ('fynbos-sweet'), maltyl isobutyrate ('caramel'), and 2-acetylpyrrole ('nutty') for honeybush.

Evaluation of emissions and exposures at workplaces using desktop 3-dimensional printer.

There is a paucity of data on additive manufacturing process emissions and personal exposures in real-world workplaces. Hence, we evaluated atmospheres in four workplaces utilizing desktop "3-dimensional" (3-d) printers [fused filament fabrication (FFF) and sheer] for production, prototyping, or research. Airborne particle diameter and number concentration and total volatile organic compound concentrations were measured using real-time instruments. Airborne particles and volatile organic compounds were collected using time-integrated sampling techniques for off-line analysis. Personal exposures for metals and volatile organic compounds were measured in the breathing zone of operators. All 3-d printers that were monitored released ultrafine and fine particles and organic vapors into workplace air. Particle number-based emission rates (#/min) ranged from 9.4 × 109 to 4.4 × 1011 (n = 9samples) for FFF3-d printers and from 1.9 to 3.8 × 109 (n = 2 samples) for a sheer 3-d printer. The large variability in emission rate values reflected variability from the printers as well as differences in printer design, operating conditions, and feedstock materials among printers. A custom-built ventilated enclosure evaluated at one facility was capable of reducing particle number and total organic chemical concentrations by 99.7% and 53.2%, respectively. Carbonyl compounds were detected in room air; however, none were specifically attributed to the 3-d printing process. Personal exposure to metals (aluminum, iron) and 12 different organic chemicals were all below applicable NIOSH Recommended Exposure Limit values, but results are not reflective of all possible exposure scenarios. More research is needed to understand 3-d printer emissions, exposures, and efficacy of engineering controls in occupational settings.

Insights Into Emissions and Exposures From Use of Industrial-Scale Additive Manufacturing Machines.

BACKGROUND: Emerging reports suggest the potential for adverse health effects from exposure to emissions from some additive manufacturing (AM) processes. There is a paucity of real-world data on emissions from AM machines in industrial workplaces and personal exposures among AM operators. METHODS: Airborne particle and organic chemical emissions and personal exposures were characterized using real-time and time-integrated sampling techniques in four manufacturing facilities using industrial-scale material extrusion and material jetting AM processes. RESULTS: Using a condensation nuclei counter, number-based particle emission rates (ERs) (number/min) from material extrusion AM machines ranged from 4.1 × 1010 (Ultem filament) to 2.2 × 1011 [acrylonitrile butadiene styrene and polycarbonate filaments). For these same machines, total volatile organic compound ERs (µg/min) ranged from 1.9 × 104 (acrylonitrile butadiene styrene and polycarbonate) to 9.4 × 104 (Ultem). For the material jetting machines, the number-based particle ER was higher when the lid was open (2.3 × 1010 number/min) than when the lid was closed (1.5-5.5 × 109 number/min); total volatile organic compound ERs were similar regardless of the lid position. Low levels of acetone, benzene, toluene, and m,p-xylene were common to both AM processes. Carbonyl compounds were detected; however, none were specifically attributed to the AM processes. Personal exposures to metals (aluminum and iron) and eight volatile organic compounds were all below National Institute for Occupational Safety and Health (NIOSH)-recommended exposure levels. CONCLUSION: Industrial-scale AM machines using thermoplastics and resins released particles and organic vapors into workplace air. More research is needed to understand factors influencing real-world industrial-scale AM process emissions and exposures.

The microbial community of a biofilm contact reactor for the treatment of winery wastewater.

AIMS: To utilize a three-tiered approach to provide insight into the microbial community structure, the spatial distribution and the metabolic capabilities of organisms of a biofilm in the two towers of a high-rate biological contact reactor treating winery wastewater. METHODS AND RESULTS: Next-generation sequencing indicated that bacteria primarily responsible for the removal of carbohydrates, sugars and alcohol were more abundant in tower 1 than tower 2 while nitrifying and denitrifying bacteria were more abundant in tower 2. Yeast populations differed in each tower. Fluorescent in situ hybridization coupled with confocal microscopy showed distribution of organisms confirming an oxygen gradient across the biofilm depth. The Biolog system (ECO plates) specified the different carbon-metabolizing profiles of the two biofilms. CONCLUSIONS: The three-tiered approach confirmed that the addition of a second subunit to the bioreactor, expanded the treatment capacity by augmenting the microbial and metabolic diversity of the system, improving the treatment scope of the system. SIGNIFICANCE AND IMPACT OF THE STUDY: A three-tiered biofilm analysis provided data required to optimize the design of a bioreactor to provide favourable conditions for the development of a microbial consortium, which has optimal waste removal properties for the treatment requirements at hand.

The Universal Anaesthesia Machine (UAM): assessment of a new anaesthesia workstation built with global health in mind.

The Universal Anaesthesia Machine has been developed as a complete anaesthesia workstation for use in low- and middle-income countries, where the provision of safe general anaesthesia is often compromised by unreliable supply of electricity and anaesthetic gases. We performed a functional and clinical assessment of this anaesthetic machine, with particular reference to novel features and functioning in the intended environment. The Universal Anaesthesia Machine was found to be reliable, safe and consistent across a range of tests during targeted functional testing.

Internal pH regulation facilitates in situ long-term acclimation of massive corals to end-of-century carbon dioxide conditions.

The resilience of tropical corals to ocean acidification depends on their ability to regulate the pH within their calcifying fluid (pHcf). Recent work suggests pHcf homeostasis under short-term exposure to pCO2 conditions predicted for 2100, but it is still unclear if pHcf homeostasis can be maintained throughout a corals lifetime. At CO2 seeps in Papua New Guinea, massive Porites corals have grown along a natural seawater pH gradient for decades. This natural gradient, ranging from pH 8.1-7.4, provides an ideal platform to determine corals' pHcf (using boron isotopes). Porites maintained a similar pHcf (~8.24) at both a control (pH 8.1) and seep-influenced site (pH 7.9). Internal pHcf was slightly reduced (8.12) at seawater pH 7.6, and decreased to 7.94 at a site with a seawater pH of 7.4. A growth response model based on pHcf mirrors the observed distribution patterns of this species in the field. We suggest Porites has the capacity to acclimate after long-time exposure to end-of-century reduced seawater pH conditions and that strong control over pHcf represents a key mechanism to persist in future oceans. Only beyond end-of-century pCO2 conditions do they face their current physiological limit of pH homeostasis and pHcf begins to decrease.

Divide and Conquer May Not Be the Optimal Approach to Retain the Desirable Estrogenic Attributes of the Cyclopia Nutraceutical Extract, SM6Met.

The genus Cyclopia, an indigenous South African fynbos plant used to prepare honeybush tea, contains phytoestrogenic compounds. An extract from C. subternata, SM6Met, displays three desirable estrogenic attributes for future development of a phytoestrogenic nutraceutical, namely, ERα antagonism, ERß agonism, and also antagonism of E2-induced breast cancer cell proliferation. Activity-guided fractionation of SM6Met was used in an attempt to isolate and identify compounds conferring the desirable estrogenic profile to SM6Met. Initial liquid-liquid fractionation of SM6Met yielded a polar fraction (PF) and a non-polar fraction (NPF), with the desirable estrogenic attributes retained in the NPF. Subsequent high performance counter-current chromatography (HPCCC) fractionation of the NPF yielded three fractions (F1-F3). Interestingly, the fractions revealed separation of the previously demonstrated positive estrogenic attributes of the NPF into separate fractions, with F1 and F2 acting as ERα antagonists, only F2 inducing antagonism of E2-induced breast cancer cell proliferation and only F3 retaining robust ERß agonist activity. In terms of major polyphenols, quantitative HPLC and liquid chromatography tandem mass spectrometry (LC-MS/MS) indicated that HPCCC fractionation resulted in a divergence of polyphenolic classes, with F1 emerging as the dihydrochalcone-rich fraction and F2 as the flavanone- and benzophenone-rich fraction, while the xanthones, flavones and phenolic acids were retained in F3. F3 was re-engineered into F3R by reassembling the major polyphenols identified in the fraction. F3R could, however, not replicate the effect of F3. In conclusion, although activity-guided fractionation results suggest that retention of all the desirable estrogenic attributes of the original SM6Met in one fraction is not an attainable goal, fractionation is a useful tool to enhance specific desirable estrogenic attributes.

Calcium dynamics in microbialite-forming exopolymer-rich mats on the atoll of Kiritimati, Republic of Kiribati, Central Pacific.

Microbialite-forming microbial mats in a hypersaline lake on the atoll of Kiritimati were investigated with respect to microgradients, bulk water chemistry, and microbial community composition. O2, H2S, and pH microgradients show patterns as commonly observed for phototrophic mats with cyanobacteria-dominated primary production in upper layers, an intermediate purple layer with sulfide oxidation, and anaerobic bottom layers with sulfate reduction. Ca(2+) profiles, however, measured in daylight showed an increase of Ca(2+) with depth in the oxic zone, followed by a sharp decline and low concentrations in anaerobic mat layers. In contrast, dark measurements show a constant Ca(2+) concentration throughout the entire measured depth. This is explained by an oxygen-dependent heterotrophic decomposition of Ca(2+)-binding exopolymers. Strikingly, the daylight maximum in Ca(2+) and subsequent drop coincides with a major zone of aragonite and gypsum precipitation at the transition from the cyanobacterial layer to the purple sulfur bacterial layer. Therefore, we suggest that Ca(2+) binding exopolymers function as Ca(2+) shuttle by their passive downward transport through compression, triggering aragonite precipitation in the mats upon their aerobic microbial decomposition and secondary Ca(2+) release. This precipitation is mediated by phototrophic sulfide oxidizers whose action additionally leads to the precipitation of part of the available Ca(2+) as gypsum.

High-dimensional nested analysis of variance to assess the effect of production season, quality grade and steam pasteurization on the phenolic composition of fermented rooibos herbal tea.

A nested analysis of variance combined with simultaneous component analysis, ASCA, was proposed to model high-dimensional chromatographic data. The data were obtained from an experiment designed to investigate the effect of production season, quality grade and post-production processing (steam pasteurization) on the phenolic content of the infusion of the popular herbal tea, rooibos, at 'cup-of-tea' strength. Specifically, a four-way analysis of variance where the experimental design involves nesting in two of the three crossed factors was considered. For the purpose of the study, batches of fermented rooibos plant material were sampled from each of four quality grades during three production seasons (2009, 2010 and 2011) and a sub-sample of each batch was steam-pasteurized. The phenolic content of each rooibos infusion was characterized by high performance liquid chromatography (HPLC)-diode array detection (DAD). In contrast to previous studies, the complete HPLC-DAD signals were used in the chemometric analysis in order to take into account the entire phenolic profile. All factors had a significant effect on the phenolic content of a 'cup-of-tea' strength rooibos infusion. In particular, infusions prepared from the grade A (highest quality) samples contained a higher content of almost all phenolic compounds than the lower quality plant material. The variations of the content of isoorientin and orientin in the different quality grade infusions over production seasons are larger than the variations in the content of aspalathin and quercetin-3-O-robinobioside. Ferulic acid can be used as an indicator of the quality of rooibos tea as its content generally decreases with increasing tea quality. Steam pasteurization decreased the content of the majority of phenolic compounds in a 'cup-of-tea' strength rooibos infusion.

Amelioration of palmitate-induced insulin resistance in C2C12 muscle cells by rooibos (Aspalathus linearis).

Increased levels of free fatty acids (FFAs), specifically saturated free fatty acids such as palmitate are associated with insulin resistance of muscle, fat and liver. Skeletal muscle, responsible for up to 80% of the glucose disposal from the peripheral circulation, is particularly vulnerable to increased levels of saturated FFAs. Rooibos (Aspalathus linearis) and its unique dihydrochalcone C-glucoside, aspalathin, shown to reduce hyperglycemia in diabetic rats, could play a role in preventing or ameliorating the development of insulin resistance. This study aims to establish whether rooibos can ameliorate experimentally-induced insulin-resistance in C2C12 skeletal muscle cells. Palmitate-induced insulin resistant C2C12 cells were treated with an aspalathin-enriched green (unfermented) rooibos extract (GRE), previously shown for its blood glucose lowering effect in vitro and in vivo or an aqueous extract of fermented rooibos (FRE). Glucose uptake and mitochondrial activity were measured using 2-deoxy-[³H]-D-glucose, MTT and ATP assays, respectively. Expression of proteins relevant to glucose metabolism was analysed by Western blot. GRE contained higher levels of all compounds, except the enolic phenylpyruvic acid-2-O-glucoside and luteolin-7-O-glucoside. Both rooibos extracts increased glucose uptake, mitochondrial activity and ATP production. Compared to FRE, GRE was more effective at increasing glucose uptake and ATP production. At a mechanistic level both extracts down-regulated PKC θ activation, which is associated with palmitate-induced insulin resistance. Furthermore, the extracts increased activation of key regulatory proteins (AKT and AMPK) involved in insulin-dependent and non-insulin regulated signalling pathways. Protein levels of the glucose transporter (GLUT4) involved in glucose transport via these two pathways were also increased. This in vitro study therefore confirms that rooibos can ameliorate palmitate-induced insulin resistance in C2C12 skeletal muscle cells. Inhibition of PKC θ activation and increased activation of AMPK and AKT offer a plausible mechanistic explanation for this ameliorative effect.

Acute assessment of an aspalathin-enriched green rooibos (Aspalathus linearis) extract with hypoglycemic potential.

Rooibos, an endemic South African plant, known for its use as herbal tea, has potential as an antidiabetic herbal product, following recent demonstration of the glucose lowering effect of its major flavonoid, the dihydrochalcone C-glucoside aspalathin. The purpose of this study was to confirm antidiabetic activity for rooibos extract high in aspalathin content. An extract (SB1) was selected after screening for high aspalathin content and α-glucosidase inhibition activity. On-line HPLC-biochemical detection confirmed α-glucosidase inhibitory activity for aspalathin. In vitro the extract induced a dose response increase in glucose uptake (5 × 10⁻5 to 5 µg/ml) on C2C12 myotubules. Aspalathin was effective at 1, 10 and 100 µM, while rutin was effective at 100 µM. In the Chang cells only the extract was effective. In vivo the extract sustained a glucose lowering effect comparable to metformin over a 6h period after administration (25mg/kg body weight (BW)) to STZ-induced diabetic rats. In an oral glucose tolerance test the extract (30 mg/kg BW) was more effective than vildagliptin (10mg/kg BW), a dipeptidyl peptidase-4 inhibitor. An aspalathin-rutin mixture (1:1; m/m) dosed at 1.4 mg/kg BW, but not the single compounds separately, reduced blood glucose concentrations of STZ-induced diabetic rats over a 6h monitoring period. The improved hypoglycemic activity of the aspalathin-rutin mixture and the extract illustrated synergistic interactions of polyphenols in complex mixtures.

An in vitro assessment of the effect of Athrixia phylicoides DC. aqueous extract on glucose metabolism.

Athrixia phylicoides DC. is an aromatic shrub indigenous to the eastern parts of Southern Africa. Indigenous communities brew "bush tea" from dried twigs and leaves of A. phylicoides, which is consumed as a beverage and used for its medicinal properties. Plant polyphenols have been shown to be beneficial to Type 2 diabetes mellitus (T2D) and obesity. Aqueous extracts of the plant have been shown to be rich in polyphenols, in particular phenolic acids, which may enhance glucose uptake and metabolism. The aim of this study was to determine the phenolic composition of a hot water A. phylicoides extract and assess its in vitro effect on cellular glucose utilisation. The most abundant phenolic compounds in the extract were 6-hydroxyluteolin-7-O-glucoside, chlorogenic acid, protocatechuic acid, a di-caffeoylquinic acid and a methoxy-flavonol derivative. The extract increased glucose uptake in C2C12, Chang and 3T3-L1 cells, respectively. Intracellular glucose was utilised by both oxidation (C2C12 myocytes and Chang cells; p < 0.01 and p < 0.05, respectively) and by increased glycogen storage (Chang cells; p < 0.05). No cytotoxicity was observed in Chang cells at the concentrations tested. The effects of the extract were not dose-dependent. A. phylicoides aqueous extract stimulated in vitro glucose uptake and metabolism, suggesting that consumption of this phenolic-rich extract could potentially ameliorate metabolic disorders related to obesity and T2D.

Niche differentiation among mat-forming, sulfide-oxidizing bacteria at cold seeps of the Nile Deep Sea Fan (Eastern Mediterranean Sea).

Sulfidic muds of cold seeps on the Nile Deep Sea Fan (NDSF) are populated by different types of mat-forming sulfide-oxidizing bacteria. The predominant sulfide oxidizers of three different mats were identified by microscopic and phylogenetic analyses as (i) Arcobacter species producing cotton-ball-like sulfur precipitates, (ii) large filamentous sulfur bacteria including Beggiatoa species, and (iii) single, spherical Thiomargarita species. High resolution in situ microprofiles revealed different geochemical settings selecting for the different mat types. Arcobacter mats occurred where oxygen and sulfide overlapped above the seafloor in the bottom water interface. Filamentous sulfide oxidizers were associated with steep gradients of oxygen and sulfide in the sediment. A dense population of Thiomargarita was favored by temporarily changing supplies of oxygen and sulfide in the bottom water. These results indicate that the decisive factors in selecting for different mat-forming bacteria within one deep-sea province are spatial or temporal variations in energy supply. Furthermore, the occurrence of Arcobacter spp.-related 16S rRNA genes in the sediments below all three types of mats, as well as on top of brine lakes of the NDSF, indicates that this group of sulfide oxidizers can switch between different life modes depending on the geobiochemical habitat setting.

Cultivation-independent characterization of 'Candidatus Magnetobacterium bavaricum' via ultrastructural, geochemical, ecological and metagenomic methods.

'Candidatus Magnetobacterium bavaricum' is unusual among magnetotactic bacteria (MTB) in terms of cell size (8-10 µm long, 1.5-2 µm in diameter), cell architecture, magnetotactic behaviour and its distinct phylogenetic position in the deep-branching Nitrospira phylum. In the present study, improved magnetic enrichment techniques permitted high-resolution scanning electron microscopy and energy dispersive X-ray analysis, which revealed the intracellular organization of the magnetosome chains. Sulfur globule accumulation in the cytoplasm point towards a sulfur-oxidizing metabolism of 'Candidatus M. bavaricum'. Detailed analysis of 'Candidatus M. bavaricum' microhabitats revealed more complex distribution patterns than previously reported, with cells predominantly found in low oxygen concentration. No correlation to other geochemical parameters could be observed. In addition, the analysis of a metagenomic fosmid library revealed a 34 kb genomic fragment, which contains 33 genes, among them the complete rRNA gene operon of 'Candidatus M. bavaricum' as well as a gene encoding a putative type IV RubisCO large subunit.

South African herbal teas: Aspalathus linearis, Cyclopia spp. and Athrixia phylicoides--a review.

Rooibos (Aspalathus linearis (Brum.f) Dahlg.) and honeybush (Cyclopia Vent. species) are popular indigenous South African herbal teas enjoyed for their taste and aroma. Traditional medicinal uses of rooibos in South Africa include alleviation of infantile colic, allergies, asthma and dermatological problems, while a decoction of honeybush was used as a restorative and as an expectorant in chronic catarrh and pulmonary tuberculosis. Traditional medicinal uses of Athrixia phylicoides DC., or bush tea, another indigenous South African plant with very limited localised use as herbal tea, include treatment of boils, acne, infected wounds and infected throats. Currently rooibos and honeybush are produced for the herbal tea market, while bush tea has potential for commercialisation. A summary of the historical and modern uses, botany, distribution, industry and chemical composition of these herbal teas is presented. A comprehensive discussion of in vitro, ex vivo and in vivo biological properties, required to expand their applications as nutraceutical and cosmeceutical products, is included, with the main emphasis on rooibos. Future research needs include more comprehensive chemical characterisation of extracts, identification of marker compounds for extract standardisation and quality control, bioavailability and identification of bio-markers of dietary exposure, investigation of possible herb-drug interactions and plant improvement with regards to composition and bioactivity.

A toxicological assessment of Athrixia phylicoides aqueous extract following sub-chronic ingestion in a rat model.

Athrixia phylicoides is an aromatic, indigenous shrub used to brew "bush tea." Surveys have shown that the consumption of bush tea is widespread and commercialization of the extract holds economic and developmental potential. Aqueous extracts of A. phylicoides are non-toxic to brine shrimp and renal cell lines; however, verification in a mammalian model is needed. In this study, daily ingestion of high doses of aqueous A. phylicoides extract by mature Wistar rats was tested for potential toxicity over a 3-month period. Three-month-old Wistar rats were randomized into a control group (receiving no extract) and three experimental groups receiving 30, 90, or 180 mg dried aqueous A. phylicoides extract/kg body mass/day. After 90 days of daily extract ingestion, blood and tissue were harvested. There was no morbidity or mortality during the study. Food and water intake, as well as body mass and stool production, were unaffected by the consumption of the extract. Urine production was increased in the 90 and 180 mg/kg groups suggesting that A. phylicoides is mildly diuretic. Serum alkaline phosphatase, creatinine, and urea levels were normal for all groups. Histopathology showed no signs of any extract induced toxicity in the liver, kidneys, gastrointestinal tract, and other tissues studied.

Determination and localisation of in situ substrate uptake by anaerobic wastewater treatment granular biofilms.

Radiotracer incubation experiments and beta microimaging, along with fluorescent in situ hybridisation (FISH), are proposed as a complementary approach to specific methanogenic activity testing and measurement of in vitro substrate utilisation rates to understand better the ecophysiology of anaerobic granular biofilms from wastewater treatment reactors.

Flowing biofilms as a transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system.

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 mum h(-1) in the turbulent flow cell and 1.0 mum h(-1) in the laminar flow cell.