Dielectrophoretic separation of Lactobacillus acidophillus bacteria from Saccharomyces cerevisiae yeasts

We show the size-based separation of model Saccharomyces cerevisiae yeast cells from a background of Lactobacillus acidophillus bacteria under the combined effect of dielectrophoretic (DEP) and hydrodynamic forces. © 2012 IEEE.

FTIR microspectroscopy for rapid screening and monitoring of polyunsaturated fatty acid production in commercially valuable marine yeasts and protists

The increase in polyunsaturated fatty acid (PUFA) consumption has prompted research into alternative resources other than fish oil. In this study, a new approach based on focal-plane-array Fourier transform infrared (FPA-FTIR) microspectroscopy and multivariate data analysis was developed for the characterisation of some marine microorganisms. Cell and lipid compositions in lipid-rich marine yeasts collected from the Australian coast were characterised in comparison to a commercially available PUFA-producing marine fungoid protist, thraustochytrid. Multivariate classification methods provided good discriminative accuracy evidenced from (i) separation of the yeasts from thraustochytrids and distinct spectral clusters among the yeasts that conformed well to their biological identities, and (ii) correct classification of yeasts from a totally independent set using cross-validation testing. The findings further indicated additional capability of the developed FPA-FTIR methodology, when combined with partial least squares regression (PLSR) analysis, for rapid monitoring of lipid production in one of the yeasts during the growth period, which was achieved at a high accuracy compared to the results obtained from the traditional lipid analysis based on gas chromatography. The developed FTIR-based approach when coupled to programmable withdrawal devices and a cytocentrifugation module would have strong potential as a novel online monitoring technology suited for bioprocessing applications and large-scale production.

Determination of astaxanthin steroisomers and colour attributes in flesh of rainbow trout (Oncorhynchus mykiss) as a tool to distinguish the dietary pigmentation source

The presence of carotenoids in animal tissue reflects their sources along the food chain. Astaxanthin, the main carotenoid used for salmonid pigmentation, is usually included in the feed as a synthetic product. However, other dietary sources of astaxanthin such as shrimp or krill wastes, algae meal or yeasts are also available on the market. Astaxanthin possesses two identical asymmetric atoms at C-3 and C-3' making possible three optical isomers with all-trans configuration of the chain: 3S,3'S, 3R,3'S, and 3R,3'R. The distribution of the isomers in natural astaxanthin differs from that of the synthetic product. This latter is a racemic mixture, with a typical ratio of 1:2:1 (3S,3'S:3R,3'S:3R,3'R), while astaxanthin from natural sources has a variable distribution of the isomers deriving from the different biological organism that synthesized it. The high-performance liquid chromatographic (HPLC) analysis of all-trans isomers of astaxanthin was performed in different pigment sources, such as red yeast Phaffia rhodozyma, alga meal Haematococcus pluvialis, krill meal and oil, and shrimp meal. With the aim to investigate astaxanthin isomer ratios in flesh of fish fed different carotenoid sources, three groups of rainbow trout were fed for 60 days diets containing astaxanthin from synthetic source, H. pluvialis algae meal and P. rhodozyma red yeast. Moreover, the distribution of optical isomers of astaxanthin in trout purchased on the Italian market was investigated. A characteristic distribution of astaxanthin stereoisomers was detected for each pigment sources and such distribution was reproduced in the flesh of trout fed with that source. Colour values measured in different sites of fillet of rainbow trout fed with different pigment sources showed no significant differences. Similarly, different sources of pigment (natural or synthetic) produced colour values of fresh fillet with no relevant or significant differences. The coefficient of distance computed amongst the feed ingredient and the trout fillet astaxanthin stereoisomers was a useful tool to identify the origin of the pigment used on farm.

Functional analysis of yeast snoRNA and snRNA 3' end formation mediated by uncoupling of cleavage and polyadenylation

Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3′-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3′ portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.

Organic compounds present in the natural Amazonian aerosol : characterization by gas chromatography–mass spectrometry

[1] As part of the Large-Scale Biosphere-Atmosphere Experiment in Amazonia (LBA)-Cooperative LBA Airborne Regional Experiment (CLAIRE) 2001 campaign in July 2001, separate day and nighttime aerosol samples were collected at a ground-based site in Amazonia, Brazil, in order to examine the composition and temporal variability of the natural “background” aerosol. We used a high-volume sampler to separate the aerosol into fine (aerodynamic diameter, AD < 2.5 μm) and coarse (AD > 2.5 μm) size fractions and quantified a range of organic compounds in methanolic extracts of the samples by a gas chromatographic-mass spectrometric technique. The carbon fraction of the compounds could account for an average of 7% of the organic carbon (OC) in both the fine and coarse aerosol fractions. We observed the highest concentrations of sugars, sugar alcohols, and fatty acids in the coarse aerosol samples, which suggests that these compounds are associated with primary biological aerosol particles (PBAP) observed in the forest atmosphere. Of these, trehalose, mannitol, arabitol, and the fatty acids were found to be more prevalent at night, coinciding with a nocturnal increase in PBAP in the 2–10 μm size range (predominantly yeasts and other small fungal spores). In contrast, glucose, fructose, and sucrose showed persistently higher daytime concentrations, coinciding with a daytime increase in large fungal spores, fern spores, pollen grains, and, to a lesser extent, plant fragments (generally >20 μm in diameter), probably driven by lowered relative humidity and enhanced wind speeds/convective activity during the day. For the fine aerosol samples a series of dicarboxylic and hydroxyacids were detected with persistently higher daytime concentrations, suggesting that photochemical production of a secondary organic aerosol from biogenic volatile organic compounds may have made a significant contribution to the fine aerosol. Anhydrosugars (levoglucosan, mannosan, galactosan), which are specific tracers for biomass burning, were detected only at low levels in the fine aerosol samples. On the basis of the levoglucosan-to-OC emission ratio measured for biomass burning aerosol, we estimate that an average of ∼16% of the OC in the fine aerosol was due to biomass burning during CLAIRE 2001, indicating that the major fraction was associated with biogenic particles.

Molecular identification of marine yeast and its spectroscopic analysis establishes unsaturated fatty acid accumulation

Marine microbes are competent organisms, some of which can accumulate large amounts of lipids. A yeast strain, Rhodotorula mucilaginosa AMCQ8A was isolated from the marine water of the Queenscliff region, Victoria, Australia. The yeast isolate was identified by sequencing 18s rDNA genes. Scanning electron microscopy images revealed scars on the surface of the yeast cells. The Fourier transform infrared spectroscopy microspectroscopy studies demonstrated the presence of unsaturated fatty acids by differential microscopic analysis. The sharp band at 1745 cm^-1 was represented by ν(C=O) stretches of ester functional groups from lipids and fats, and therefore indicated the presence of total lipids produced by the cells. Over 65% of the fatty acids from the yeast strain were analyzed as C¹⁶ and C^18:1 with omega-3 content from about 6% to 7%. Thus, this marine-derived yeast could be a potential source of lipids, including omega-3 fatty acids. 2012, The Society for Biotechnology, Japan. All rights reserved.

Isolation and characterisation of omega-3 fatty acids producing marine microbes

As a renewable alternative to fish oil, microbial-derived omega-3-fatty acids can be potential nutritional supplements. This study reported the isolation of novel oleaginous marine microbes from the Victorian marine environment capable of producing omega-3-fatty acids and other bio-actives. Fermentation strategies using low cost substrates improved omega-3-oils production in selected isolates.

Evaluation of bread crumbs as a potential carbon source for the growth of Thraustochytrid species for oil and omega-3 production

The utilization of food waste by microorganisms to produce omega-3 fatty acids or biofuel is a potentially low cost method with positive environmental benefits. In the present study, the marine microorganisms Thraustochytrium sp. AH-2 and Schizochytrium sp. SR21 were used to evaluate the potential of breadcrumbs as an alternate carbon source for the production of lipids under static fermentation conditions. For the Thraustochytrium sp. AH-2, submerged liquid fermentation with 3% glucose produced 4.3 g/L of biomass and 44.16 mg/g of saturated fatty acids after seven days. Static fermentation with 0.5% and 1% breadcrumbs resulted in 2.5 and 4.7 g/L of biomass, and 42.4 and 33.6 mg/g of saturated fatty acids, respectively. Scanning electron microscopic (SEM) studies confirmed the growth of both strains on breadcrumbs. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy for both strains were consistent with the utilization of breadcrumbs for the production of unsaturated lipids, albeit at relatively low levels. The total lipid yield for static fermentation with bread crumbs was marginally lower than that of fermentation with glucose media, while the yield of unsaturated fatty acids was considerably lower, indicating that static fermentation may be more appropriate for the production of biodiesel than for the production of omega-3 rich oils in these strains.

Challenges and advances in the heterologous expression of cellulolytic enzymes: a review

Second generation biofuel development is increasingly reliant on the recombinant expression of cellulases. Designing or identifying successful expression systems is thus of preeminent importance to industrial progress in the field. Recombinant production of cellulases has been performed using a wide range of expression systems in bacteria, yeasts and plants. In a number of these systems, particularly when using bacteria and plants, significant challenges have been experienced in expressing full-length proteins or proteins at high yield. Further difficulties have been encountered in designing recombinant systems for surface-display of cellulases and for use in consolidated bioprocessing in bacteria and yeast. For establishing cellulase expression in plants, various strategies are utilized to overcome problems, such as the auto-hydrolysis of developing plant cell walls. In this review, we investigate the major challenges, as well as the major advances made to date in the recombinant expression of cellulases across the commonly used bacterial, plant and yeast systems. We review some of the critical aspects to be considered for industrial-scale cellulase production.

Propyl gallate and butylated hydroxytoluene influence the accumulation of saturated fatty acids, omega-3 fatty acid and carotenoids in thraustochytrids

Schizochytrium sp. S31 was shown to have potential for production of the functional food ingredients docosahexaenoic acid (DHA) and astaxanthin, with coproduction of biodiesel. Biomass and lipid levels were greater with glycerol than with glucose as carbon source. Addition of propyl gallate or butylated hydroxytoluene to the media resulted in increased biomass and lipid levels, with propyl gallate being the more effective of the two antioxidants. Medium supplementation with propyl gallate at 0.03% and glycerol as the carbon source resulted in enhanced biomass productivity (28.50 g L^-1), lipid accumulation (24.87 g L^-1) and astaxanthin levels (452.26 μg L^-1).