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Purpose A large proportion of the European Union’s tomato crop is discarded during harvesting and there is a valorisation potential to recover proteins from this waste. Methods Cherry tomatoes were segregated into three separate components: juice, pomace (peels and skins), and seeds. The peels and skins, and seeds were separately hydrolyzed with carbohydrases to determine whether protein recovery could be increased. In addition, a strategy to fractionate the seeds was developed using sequential washing of milled tomato seeds, followed by low-speed centrifugation to remove the denser seed hulls and to collect the protein rich kernels remaining in suspension. Results The protein content of the seeds was highest with 27.4% while the peels and skins contained 7.6%. Carbohydrase mediated hydrolysis resulted in a minor increase in protein recovery of 10% from seeds using Filta 02L (cellulase, xylanase and β-glucanase), and the quantity of protein recovered from peels and skins increased by 210% using Tail 157 (pectinase, hemicellulase). The strategy to separate the seeds into two fractions, revealed that a higher proportion of the fibre (65%) was associated with the hull fraction compared with the original seeds (47%). A significant proportion of the fibre in this fraction was composed of lignin although the protein contents between both fractions was similar ranging from 27.4 to 29.9%. Conclusions These results reveal that carbohydrases were quite effective in protein extraction from peels and skins, but not from seeds. An alternative strategy was developed to remove the seed hulls from the milled seeds and to collect a fraction containing protein and dietary fibre where oil could be removed at this stage. Graphical Abstract

PurposeA large proportion of the European Union’s tomato crop is discarded during harvesting and there is a valorisation potential to recover proteins from this waste. MethodsCherry tomatoes were segregated into three separate components: juice, pomace (peels and skins), and seeds. The peels and skins, and seeds were separately hydrolyzed with carbohydrases to determine whether protein recovery could be increased. In addition, a strategy to fractionate the seeds using sequential washing of milled tomato seeds followed by low-speed centrifugation to remove the denser seed hulls and to collect the protein rich kernels remaining in suspension. ResultsThe protein content of the seeds was highest with 27.4% while the peels and skins contained 7.6%. Carbohydrase mediated hydrolysis revealed a minor increase in protein recovery from seeds by 10% using Filta 02L (cellulase, xylananse and β-glucanase), and the quantity of protein recovered from peels and skins increased by 210% using Tail 157 (pectinase, hemicellulase). The strategy to separate the seeds into two fractions, revealed that a higher proportion of the fibre (65%) was associated with the hull fraction compared with the original seeds (47%). A significant proportion of the fibre in this fraction was composed lignin although the protein contents between both fractions was similar ranging from 27.4% to 29.9%. ConclusionsThese results reveal that carbohydrases were quite effective in protein extraction from peels and skins, but not from seeds. An alternative strategy was developed to remove the seed hulls from the milled seeds and to collect a crude protein fraction where the protein content could be further improved.

The conversion of raw fruits and vegetables, including tomatoes into processed food products creates side streams of residues that can place a burden on the environment. However, these processed residues are still rich in bioactive compounds and in an effort to valorize these materials in tomato by-product streams, the main aim of this study is to extract proteins and identify the main phenolic compounds present in tomato pomace (TP), peel and skins (TPS) by HPLC-DAD-ESI-QTOF. Forty different phenolic compounds were identified in the different tomato extracts, encompassing different groups of phenolic compounds, including derivatives of simple phenolic acid derivatives, hydroxycinnamoylquinic acid, flavones, flavonones, flavonol, and dihydrochalcone. In the crude protein extract (TPE) derived from tomatoes, most of these compounds were still present, confirming that valuable phenolic compounds were not degraded during food processing of these co-product streams. Moreover, phenolic compounds present in the tomato protein crude extract could provide a valuable contribution to the required daily intake of phenolics that are usually supplied by consuming fresh vegetables and fruits.

The application of solid-state fermentation offers an alternative to conventional, submerged approaches for a variety of bioconversion processes, including animal feeds, biofuels and fungal bioproducts. Optimizing solid-state fermentation under low moisture conditions could significantly impact the proportion of dry biomass that could be processed and improve the commercial viability of this approach, because of reduced input costs and higher yields of final products. Pleurotus erygnii that appeared to show tolerance to low moisture conditions was grown on saturated and desaturated wheat straw. Pleurotus erygnii showed insignificant fibre degradation although showed significantly lower biomass decomposition on desaturated wheat straw. Fibre decomposition by the fungus on wheat straw containing wheat bran showed marginally higher decomposition when saturated although there was no difference in biomass decomposition. The levels of delignification achieved were similar under different saturation conditions. It would appear that the fungus effectively decomposed fibre under low moisture conditions often resulting in lower biomass losses. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a white rot fungus, Pleurotus erygnii, effectively decomposed fibre under low moisture conditions when grown on wheat straw at similar levels under higher moisture conditions. However, the addition of wheat bran to wheat straw created a heterogeneous system that appeared to allow P. erygnii to thrive under much lower moisture conditions although lower levels of fibre decomposition was obtained. These factors could influence the preparation of solid-state fermentation.

Graphical abstract, Highlights • In-line electroanalysis of d-glucose during bioethanol production. • Second-generation biosensor suitable for industrial process analytical technology. • Greater d-glucose yield from wheat straw refined under pressure. • d-Glucose productivity is 20–40 mg/(L h) over a 5 L scale., Mechanical pre-treatment (disc refining) of wheat straw, at both atmospheric and elevated pressure, is shown to be an efficient process to access fermentable monosaccharides, with the potential to integrate within the infrastructure of existing first-generation bioethanol plants. The mild, enzymatic degradation of this sustainable lignocellulosic biomass affords ca. 0.10–0.13 g/g (dry weight) of d-glucose quantifiable voltammetrically in real time, over a two hundred-fold range in experimental laboratory scales (25 mL to 5.0 L), with pressure disc refining of the wheat straw enabling almost twice the amount of d-glucose to be generated during the hydrolysis stage than experiments using atmospheric refining (0.06–0.09 g/g dry weight). Fermentation of the resulting hydrolysate affords 0.08–0.10 g/g (dry weight) of ethanol over similar scales, with ethanol productivity at ca. 37 mg/(L h). These results demonstrate that minimal cellulose decomposition occurs during pressure refining of wheat straw, in contrast to hemicellulose, and suggest that the development of green, mechanochemical processes for the scalable and cost-effective manufacture of second-generation bioethanol requires improved cellulose decomposition.

The valorization of olive pomace through the extraction of phenolic compounds at an industrial scale is influenced by several factors that can have a significant impact on the feasibility of this approach. These include the types and levels of phenolic compounds that are present, the impact that seasonal variation and cultivar type have on the phenolic compound content in both olive pomace and mill effluents and the technological approach used to process the olive crop. Chemical analysis of phenolic compounds was performed using an HPLC-diode-array detector (DAD)-qTOF system, resulting in the identification of 45 compounds in olive mill wastewater and pomace, where secoiridoids comprised 50&ndash, 60% of the total phenolic content. This study examined three different factors that could impact the phenolic compound content of these processing streams, including cultivar types typically grown on local farms in Slovenia, the type of downstream processing used and seasonality effects. Olive crop varieties sourced from local farms showed high variability, and the highest phenolic content was associated with the local variety &ldquo, Istrska Belica&rdquo, During processing, the phenolic content was on average approximately 50% higher during two-phase decanting compared to three-phase decanting and the type of compound present significantly different. An investigation into the seasonal effects revealed that the phenolic content was 20% higher during the 2019 growing season compared to 2018. A larger sample size over additional growing seasons is required to fully understand the annual variation in phenolic compound content. The methods and results used in this study provide a basis for further analysis of phenolic compounds present in the European Union&rsquo, s olive crop processing residues and will inform techno-economic modelling for the development of olive biorefineries in Slovenia.

The agricultural production of olives, rapeseed, tomatoes and citrus fruits within Europe is significant, resulting in a considerable amount of residual material. Rapeseed contains a high proportion of protein but the presence of anti-nutritional components, including glucosinolates, limits its use in food and feed applications. In contrast, the protein quantities associated with the other crop residues are much lower, although each of the residues could be separated into different constitutive parts where some have shown higher protein contents. A variety of different enzymatic based approaches to deconstruct crop residues have shown to be effective in increasing the yields of protein recovered. These studies show that valorisation of selected crop components could form the basis of a crop biorefinery process to capture proteins and other potentially useful compounds.

This preliminary study assesses a potential pre-treatment approach for agricultural residues, in order to improve enzyme access and cellulose digestibility that increased with increasing refining pressure. Wheat straw, an important European agri-residue, was chopped then refined at pilot-scale under different pressures (4–10 bar) and two refiner plate configurations. The most energy efficient runs used 0.94–0.96 kWh electricity; 8.9–11.0 MJ heat per kg dry matter fibre. A scaling factor specific to the machinery used in the trial suggested that wheat straw could be refined using approximately 160 kWh electricity and 980–1900 MJ heat per tonne DM yield at commercial-scale. Hemicellulose content in wheat straw at 31.8% decreased to the lowest level of 14.6% after refining at 10 bar. Pressurised disc refining did not appear to produce significant quantities of acetic acid, a key fermentation inhibitor, that could limit microbial fermentation.

The function of packaging is crucial in the maintenance of fresh meat product quality. This study aimed to assess the efficiency of six films added with coatings 2379L/220 and 2379L/221 (containing sage extracts) to inhibit Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli, which showed that two of the six films had a significant effect. Additionally, the effects of the films on refrigerated skinless chicken breast meat were evaluated based on microbiological content, colour, weight loss, texture and pH. Four of the six films were examined could extend the storability of refrigerated chicken breast fillets for up to seven days. All six treated films improved the pH, colour stability, weight loss, and texture of the chicken fillets. Therefore, these findings suggested that the coatings containing sage extracts having different viscosities (2379L/220 and 2379L/221) were effective as antimicrobial adhesives in food packaging films and can be commercially applied in prolonging the storage of chicken breast meat without affecting their quality.

The fungal enzyme activities and breakdown of wood components may be influenced by the fibrous and structural composition across different wood species. White-rot fungi were inoculated onto wood chips of F. excelsior, A. pseudoplatanus or Q. robur and incubated for 28 days revealing that most fungi appeared to successfully colonize the different types of wood chips. Fibre analysis revealed that the fungi causing the highest mass losses in F. excelsior and A. pseudoplatanus were those that degraded more cellulose compared with hemicellulose or lignin. Fungal degradation leading to high mass losses of Q. robur was more complicated as lignin-degrading activities became more important. The structural composition in terms of the largest vessel sizes only showed an inverse correlation with remaining moisture content and not with mass loss or fibre degradation. These results provide an insight into fungal degradation of wood from three common tree species, and the link between the compositional characteristics of each wood type and the ease of degradation. This could have an impact on future biological pre-treatment strategies and valorisation approaches for forestry residues in integrated biorefineries.

Microorganisms are the primary agents responsible for the modification, degradation, and/or detoxification of pollutants, and thus, they play a major role in their natural attenuation; yet, little is known about the structure and diversity of the subsurface community and relationships between microbial community and groundwater hydrochemistry. In this study, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) allowed a comparative microbial community analysis of sulfate-contaminated groundwater samples from nine different wells in the region of Baogang rare earth tailings. Using real-time PCR, the abundance of total bacteria and the sulfate-reducing genes of aprA and dsrB were quantified. Statistical analyses showed a clear distinction of the microbial community diversity between the contaminated and uncontaminated samples, with Proteobacteria being the most dominant members of the microbial community. SO4 2− concentrations exerted a significant effect on the variation of the bacterial community (P

Pressure refining, a pulp making process to separate fibres of lignocellulosic materials, deposits lignin granules on the surface of the fibres that could enable increased access to lignin degrading enzymes. Three different white rot fungi were grown on pressure refined (at 6 bar and 8 bar) and milled Miscanthus. Growth after 28 days showed highest biomass losses on milled Miscanthus compared to pressure refined Miscanthus. Ceriporiopsis subvermispora caused a significantly higher proportion of lignin removal when grown on 6 bar pressure refined Miscanthus compared to growth on 8 bar pressure refined Miscanthus and milled Miscanthus. RM22b followed a similar trend but Phlebiopsis gigantea SPLog6 did not. Conversely, C. subvermispora growing on pressure refined Miscanthus revealed that the proportion of cellulose increased. These results show that two of the three white rot fungi used in this study showed higher delignification on pressure refined Miscanthus than milled Miscanthus.

Several different methods were compared to recover a significant quantity of high quality RNA from H. simulans containing RNA of marine-derived fungi. Further processing of the RNA showed that amplification products could be obtained using fungal specific primers, RNA enriched in mRNA could be recovered and an oligonucleotide-linker could be attached to cDNA. The potential applications of these methods are discussed for future studies in determining whether marine fungi could be symbiotically associated with marine sponges.

Assessing metal bioavailability in soil is important in modeling the effects of metal toxicity on the surrounding ecosystem. Current methods based on diffusive gradient thin films (DGTs) and Gel-Integrated Microelectrode are limited in their availability and sensitivity. To address this, Shewanella oneidensis, an anaerobic iron reducing bacterium, was incorporated into a thin layer of agarose to replace the polyacrylamide gel that is normally present in DGT to form biologically mobilizing DGT (BMDGT). Viability analysis revealed that 16-35% of the cells remained viable within the BMDGTs depending on the culturing conditions over a 20 h period with/without metals. Deployment of BMDGTs in standardized metal solutions showed significant differences to cell-free BMDGTs when cells grown in Luria Broth (LB) were incorporated into BMDGTs and deployed under anaerobic conditions. Deployment of these BMDGTs in hematite revealed no significant differences between BMDGTs and BMDGTs containing heat killed cells. Whether heat killed cells retain the ability to affect bioavailability is uncertain. This is the first study to investigate how a microorganism that was incorporated into a DGT device such as the metal reducing bacteria, S. oneidensis, may affect the mobility of metals.

Aims: The conversion of cheap cellulosic biomass to more easily fermentable sugars requires the use of costly cellulases. We have isolated a series of marine sponge-derived fungi and screened these for cellulolytic activity to determine the potential of this unique environmental niche as a source of novel cellulase activities. Methods and Results: Fungi were isolated from the marine sponge Haliclona simulans. Phylogenetic analysis of these and other fungi previously isolated from H. simulans showed fungi from three phyla with very few duplicate species. Cellulase activities were determined using plate-based assays using different media and sea water concentrations while extracellular cellulase activities were determined using 3,5-dinitrosalicylic acid (DNSA)-based assays. Total and specific cellulase activities were determined using a range of incubation temperatures and compared to those for the cellulase overproducing mutant Hypocrea jecorina QM9414. Several of the strains assayed produced total or relative endoglucanase activities that were higher than H. jecorina, particularly at lower reaction temperatures. Conclusions: Marine sponges harbour diverse fungal species and these fungi are a good source of endoglucanase activities. Analysis of the extracellular endoglucanase activities revealed that some of the marine-derived fungi produced high endoglucanase activities that were especially active at lower temperatures. Significance and Impact of the Study: Marine-derived fungi associated with coastal marine sponges are a novel source of highly active endoglucanases with significant activity at low temperatures and could be a source of novel cellulase activities.

The diversity and antimicrobial activities of 80 fungi isolated from Haliclona simulans were assessed using different fungal media containing either agar or gellum gum. In total, 19 different genotypes were detected. These fungal isolates could be classified as members of the Agaricomycotina, Mucoromycotina, Saccharomycotina, and Pezizomycotina, although the majority of the isolates were associated with the latter class. Some of these fungal isolates showed antimicrobial inhibition of Escherichia coli, Bacillus sp., Staphylococcus aureus, and Candida glabrata. Fungal 18S rRNA gene sequences belonging to Eurotiales, Calosphaeriales, and Chaetothyriales were amplified from DNA and RNA extracted from this marine sponge. This study indicates that in contrast to the low diversity of fungi detected by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR amplification from extracts of this marine sponge, a much higher diversity of fungi could be cultured. The data suggests that some fungi live in symbiosis with H. simulans, whereas other fungi may have been ingested from the surrounding seawater.

Isolates from the Mir space station identified as Pseudomonas sp. and Stenotrophomonas maltophilia were subjected to clinorotation to model reduced gravity conditions in water in slow turning lateral vessels (STLVs). To examine cells in varying physiological states, bacteria were enumerated based on the Live/Dead Bac Light kit, DAPI (4′,6-diamidino-2-phenylindole) staining, fluorescent in situ hybridization (FISH), and colony forming units (CFU). Both Pseudomonas sp. and S. maltophilia showed a slight increase in abundance over time but only cells of Pseudomonas sp. were affected by modeled reduced gravity. For Pseudomonas sp. numbers of DAPI stained cells were significantly higher under modeled reduced gravity compared to normal gravity. In addition, the abundance of cells attached to stainless steel disks, on one sampling date, was greater for the Pseudomonas isolate under modeled reduced gravity than normal gravity. The isolates examined did not appear to appreciably enter into a viable, but not culturable state during the experiments. In general, differences between treatments were not great, demonstrating that responses to reduced gravity are less apparent under starvation conditions, compared to earlier studies which used more rich nutrient sources.

AIMS: Bacteria are important residents of water systems, including those of space stations which feature specific environmental conditions, such as lowered effects of gravity. The purpose of this study was to compare responses with modelled reduced gravity of space station, water system bacterial isolates with other isolates of the same species. METHODS AND RESULTS: Bacterial isolates, Stenotrophomonas paucimobilis and Acinetobacter radioresistens, originally recovered from the water supply aboard the International Space Station (ISS) were grown in nutrient broth under modelled reduced gravity. Their growth was compared with type strains S. paucimobilis ATCC 10829 and A. radioresistens ATCC 49000. Acinetobacter radioresistens ATCC 49000 and the two ISS isolates showed similar growth profiles under modelled reduced gravity compared with normal gravity, whereas S. paucimobilis ATCC 10829 was negatively affected by modelled reduced gravity. CONCLUSIONS: These results suggest that microgravity might have selected for bacteria that were able to thrive under this unusual condition. These responses, coupled with impacts of other features (such as radiation resistance and ability to persist under very oligotrophic conditions), may contribute to the success of these water system bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Water quality is a significant factor in many environments including the ISS. Efforts to remove microbial contaminants are likely to be complicated by the features of these bacteria which allow them to persist under the extreme conditions of the systems.

Four bacterial isolates (Chryseobacterium sp., Pseudomonas fluorescens and two Stenotrophomonas maltophilia isolates) originally isolated from the water system aboard the Mir Space Station were grown in two concentrations of nutrient broth under modeled reduced gravity using clinorotation. Sampling was performed over a 7 day period and planktonic cells were enumerated using 4′,6-diamidino-2-phenylindole (DAPI), while those attached to stainless steel were enumerated using the LIVE/DEAD® BacLight™ kit and DAPI. On some of the sampling days for all the isolates, planktonic cell counts were higher under modeled reduced gravity as compared with the normal gravity controls. In contrast, the number of cells of P. fluorescens and one S. maltophilia isolate attached to the stainless steel disks was higher under modeled reduced gravity as compared with normal gravity, whereas no such differences were observed for Chryseobacterium sp. and the other S. maltophilia isolate. Differences in motility among isolates appeared to influence the growth of planktonic cells under modeled reduced gravity but did not appear to be related to biofilm formation.

Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

Bacteria and virus-like particles (VLP) were enumerated in water, leaf, and sediment samples collected seasonally from the West Branch of the Mahoning River (OH, USA). In water, bacteria ranged from 9.37-46.5 x 10 5 cells per ml, VLP were 1.97-5.68 x 10 6 per ml, and virus: bacteria ratios (VBRs) were 0.42-3.49. In leaf samples, bacteria were 2.09-49.5 x 10 8 per g, VLP were 4.81-21.8 x 10 8 per g, and VBRs were 0.40-2.55. In sediment samples, bacteria were 1.85-7.07 x 10 7 per g, VLP were 4.71-8.91 x 10 6 per g and VBRs were 0.09-0.45. Bacterial numbers in water were comparable to previous studies and were highest in spring. Occasionally, VLP numbers in water were significantly higher than bacterial counts. In contrast, VLP numbers were often lower than bacterial numbers in leaf and sediment samples and did not vary as much among dates. Only in the leaf samples was there a significant linear relationship between VLP and bacterial abundance based on regression analysis. Compared to lakes and marine systems, VLP abundance was low in this system and a virus-bacteria relationship was generally not apparent. In lotic ecosystems, rapid changes in environmental conditions may result in a less direct virus-bacteria connection than in other aquatic ecosystems.

Ennoblement, a phenomenon in which open-circuit potential is elevated to a noble value, triggers metal corrosion in the environment and is considered to be biologically catalysed. This study investigated the involvement of marine microorganisms in the ennoblement of stainless steel coupons in sea water pumped from Kamaishi Bay. Scanning electron microscopy (SEM) showed significant attachment of prosthecate bacteria on the surfaces of stainless steel coupons in the course of ennoblement. In denaturing gradient gel electrophoresis (DGGE) analyses of polymerase chain reaction-amplified bacterial 16S rDNA fragments, several major bands were detected from the surface of the ennobled coupons, including those affiliated with the alpha and gamma subclasses of the Proteobacteria. After these observations, bacterial strains were isolated from the surface of the ennobled coupon. The 16S rDNA analysis revealed that a bacterial isolate (designated PWB3) corresponded to a major DGGE band representing an alpha-Proteobacterial population; a database analysis showed that its closest relative was Rhodobium spp., albeit with low homology ( approximately 89%). SEM indicated that this bacterium was a prosthecate bacterium that was morphologically similar to those observed on the ennobled coupons. In pure culture of strain PWB3, stainless steel coupons were ennobled when the culture was supplemented with MnCl2. Manganese was recovered from the surface of the ennobled coupons after treatment with a reducing agent. These results suggest that the attachment of manganese-oxidizing prosthecate bacteria triggered the ennoblement of stainless steel in Kamaishi Bay sea water.

The particulate methane monooxygenase genes (pMMO) were amplified from methane-biostimulated aquifer samples using two sets of primers, A189 plus A682 and pmof2 plus pmor, and the products were cloned and sequenced. The analysis of the sequences revealed a high diversity of pMMO genes in the aquifers. Some of the pMMO gene sequences (LP20 and LP21) isolated in this study were unique and unrelated to methanotrophs previously isolated from this site. Throughout all the samples collected from the different methane biostimulation trials, competitive PCR amplification using specific pMMO primers revealed that type I methanotrophs predominated over type II methanotrophs. The partial soluble methane monooxygenase genes were also amplified from the aquifer samples using specially designed primers but sequencing revealed a lower diversity.

Purpose: To determine the efficacy of (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylic acid (OTZ) in reducing urinary oxalate excretion under hyperoxaluric conditions and to determine whether by inclusion of glycolate in a standard diet, cysteine:glyoxylate adduct can be detected in hyperoxaluric rats given either compound.Materials and Methods: Hyperoxaluria (200% above basal) was induced 2 days prior to commencement of the studies and maintained throughout. After a 3 days baseline, animals were randomly allocated to a control or treatment group. Standard diet containing either (L)-cysteine or OTZ was then fed to the treatment groups for 5 days while standard diet alone was fed to the control groups. Urinary oxalate excretion was subsequently monitored average daily rates were then compared with basal values. Plasma and urine were analyzed for adduct.Results: Both (L)-cysteine and OTZ significantly reduced urinary oxalate excretion relative to the basal hyperoxaluric level (28.6 +/− 1.5 micro ...

A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.

Purpose: To determine whether (D)-penicillamine is effective in reducing hepatic oxalate production and urinary oxalate excretion.Materials and Methods: (D)-Penicillamine was administered orally to rats to determine its effect on urinary oxalate excretion and used in isolated rat hepatocytes to investigate the effect of (D)-penicillamine on oxalate production from glycolate. Studies involving hepatic aminotransferases and hepatocytes isolated from (D)-penicillamine treated rats were used to clarify the discrepancy between the in vitro and in vivo results.Results: In hepatocytes (D)-penicillamine lead to a significant reduction in oxalate production from glycolate. In vivo however, (D)-penicillamine led to a significant increase in urinary oxalate excretion and a decrease in plasma aminotransferase activity. Hepatic aminotransferases are involved in diverting oxalate precursors from oxalate production. In vitro, (D)-penicillamine was shown to inhibit hepatic aminotransferases. Hepatocytes isolated ...

The effects of orally administered (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylate (OTC) on urinary oxalate excretion were investigated in male Porton rats, as (L)-cysteine has been shown to form an adduct with glyoxylate in vitro. Feeding of OTC (204 +/- 1 mg. per day) for 5 days increased urinary cyst(e)ine, p0.001; sulphate, p0.001; phosphate, p0.05; and calcium, p0.05; and decreased urinary pH, p0.001. In addition, OTC feeding significantly decreased urinary oxalate excretion when compared with controls, p0.05 (delta OTC-delta Control -4.26 +/- 1.55 nmol./day/gm.). In the 5-day period after cessation of OTC feeding, all urinary parameters returned to control levels. At the completion of this recovery period there were no significant differences in any of the clinically significant plasma parameters. When fed for 22 days (191 +/- 3 mg. per day) OTC decreased urinary oxalate compared with controls, p0.05 (delta OTC-delta Control -9.47 +/- 4.24 nmol./day/gm.). Other urinary parameters (uric acid, magnesium, calcium, phosphate, creatinine, pH and volume) were not significantly altered by OTC feeding. Again, at the completion of this feeding period there were no significant differences in any of the clinically significant plasma parameters. (L)-cysteine feeding for 5 days (184 +/- 10 mg. per day) increased urinary sulphate, p0.001; and magnesium, p0.05, and decreased urinary pH, p0.001. In addition, (L)-cysteine feeding did not significantly change urinary oxalate excretion when compared with the controls (delta(L)-Cysteine-delta Control -2.94 +/- 2.14 nmol./day/gm.). However, at the completion of this feeding period, plasma urate, p0.02; and glucose, p0.05, were decreased, and plasma potassium, p0.01, was increased. these results indicate that orally administered OTC is effective in reducing urinary oxalate excretion without altering plasma biochemistry. It is suggested that (L)-cysteine-glyoxylate adduct formation is the mechanism by which OTC reduces urinary oxalate excretion through a reduction in endogenous oxalate production.

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.

To determine whether in patients with an ambulance response time of5min who were in VF cardiac arrest, 3min of CPR before the first defibrillation was more effective than immediate defibrillation in improving survival to hospital discharge.This randomised control trial was run by the South Australian Ambulance Service between 1 July, 2005, and 31 July, 2007. Patients in VF arrest were eligible for randomisation. Exclusion criteria were: (i)18 years of age, (ii) traumatic arrest, (iii) paramedic witnessed arrest, (iv) advanced life support performed before arrival of paramedics and (v) not for resuscitation order or similar directive. The primary outcome was survival to hospital discharge with secondary outcomes being neurological status at discharge, the rate of return of spontaneous circulation (ROSC) and the time from first defibrillation to ROSC.For all response times, no differences were observed between the immediate defibrillation group and the CPR first group in survival to hospital discharge (17.1% [18/105] vs. 10.3% [10/97]; P=0.16), the rate of ROSC (53.3% [56/105] vs. 50.5% [49/97]; P=0.69) or the time from the first defibrillation to ROSC (12:37 vs. 11:19; P=0.49). There were also no differences between the immediate defibrillation group and the CPR first group, for response times ofor = or5min: survival to hospital discharge (50.0% [7/14] vs. 25.0% [4/16]; P=0.16 or 12.1% [11/91] vs.7.4% [6/81]; P=0.31, respectively) and the rate of ROSC (71.4% [10/14] vs. 75.0% [12/16]; P=0.83 or 50.5% [46/91] vs. 45.7% [37/81]; P=0.54, respectively). No differences were observed in the neurological status of those surviving to hospital discharge.For patient in out-of-hospital VF cardiac arrest we found no evidence to support the use of 3min of CPR before the first defibrillation over the accepted practice of immediate defibrillation.

The effects of simulated microgravity on two bacterial isolates, Sphingobacterium thalpophilium and Ralstonia pickettii (formerly Burkholderia pickettii), originally recovered from water systems aboard the Mir space station were examined. These bacteria were inoculated into water, high and low concentrations of nutrient broth and subjected to simulated microgravity conditions. S. thalpophilium (which was motile and had flagella) showed no significant differences between simulated microgravity and the normal gravity control regardless of the method of enumeration and medium. In contrast, for R. pickettii (that was non-motile and lacked flagella), there were significantly higher numbers in high nutrient broth under simulated microgravity compared to normal gravity. Conversely, when R. pikkettii was inoculated into water (i.e., starvation conditions) significantly lower numbers were found under simulated microgravity compared to normal gravity. Responses to microgravity depended on the strain used (e.g., the motile strain exhibited no response to microgravity, while the non-motile strain did), the method of enumeration, and the nutrient concentration of the medium. Under oligotrophic conditions, non-motile cells may remain in geostationary orbit and deplete nutrients in their vicinity, while in high nutrient medium, resources surrounding the cell may be sufficient so that high growth is observed until nutrients becoming limiting.

Soil core samples were obtained from a trichloroethylene (TCE)-contaminated aquifer before and after the start of methane biostimulation. DNA was extracted directly from the soil samples, and denaturing gradient gel electrophoresis (DGGE) was used to analyse bacterial 16S ribosomal DNA fragments that were PCR amplified from these DNA samples. This analysis consistently detected two phylotypes in the methane-injected samples. These phylotypes were closely related to Methylobacter and Methylomonas, both belonging to type I methanotrophs. A competitive DGGE analysis using Methylosinus trichosporium OB3b cells as an internal quantitative standard showed that these populations accounted for 10(8)-10(9) cells g(-1) soil. These results showed that type I methanotrophs formed a significant proportion of the bacterial community during methane biostimulation. The implications of this finding for TCE bioremediation were discussed.

The development of molecular microbial ecology in the 1990s has allowed scientists to realize that microbial populations in the natural environment are much more diverse than microorganisms so far isolated in the laboratory. This finding has exerted a significant impact on environmental biotechnology, since knowledge in this field has been largely dependent on studies with pollutant-degrading bacteria isolated by conventional culture methods. Researchers have thus started to use molecular ecological methods to analyze microbial populations relevant to pollutant degradation in the environment (called environmentally relevant microorganisms, ERMs), although further effort is needed to gain practical benefits from these studies. This review highlights the utility and limitations of molecular ecological methods for understanding and advancing environmental biotechnology processes. The importance of the combined use of molecular ecological and physiological methods for identifying ERMs is stressed.

Despite the great effort that has gone into investigating urolithiasis, this condition still persists as one of the major ailments of the urinary tract. Calcium oxalate urolithiasis is the most common form, accounting for some 60 to 80% of total stones. This review examines the elements (i.e., urine volume and pH and urinary excretion of calcium, oxalate, citrate, urate, magnesium, pyrophosphate, and glycosaminoglycans) that give rise to idiopathic calcium oxalate urolithiasis. Treatment strategies for idiopathic calcium oxalate urolithiasis, including lithotripsy, also are discussed. Urinary oxalate excretion is a major risk factor for calcium oxalate urolithiasis, with 85 to 95% of the urinary load derived endogenously. The factors controlling endogenous oxalate production are reviewed, including pathways for the diversion of glyoxylate from oxalate production. The use of beta-aminothiols and other substances to reduce endogenous oxalate production in subjects with idiopathic calcium oxalate urolithiasis is also discussed. A review of current methodologies for the determination of urinary oxalate is also included.