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Alcohol Availability and Onset and Recurrence of Alcohol Use Disorder: Examination in a Longitudinal Cohort with Cosibling Analysis.

BACKGROUND: Recent reviews of associations of alcohol availability with alcohol outcomes suggest findings are highly inconsistent and highlight a lack of longitudinal and causal evidence. Effect modification (moderation or statistical interaction), which could contribute to the inconsistent picture in the existing literature, has not been systematically assessed. We examined associations of alcohol availability with onset and recurrence of alcohol use disorder (AUD) using multilevel, longitudinal population data from Sweden and tested hypothesized effect modifiers to identify groups for whom increased alcohol availability may be particularly risky. We also employed cosibling models to assess potential causality for AUD onset by accounting for genetic and shared-environment confounders. METHODS: Data come from all individuals born in Sweden between 1950 and 1975 who were registered in a residential neighborhood at the end of 2005 (N = 2,633,922). We used Cox proportional hazards models to investigate time to AUD onset and logistic regression to assess the odds of AUD recurrence over an 8-year period. RESULTS: Living in a neighborhood with at least 1 alcohol outlet of any type was associated with a small increase in the likelihood of developing AUD, with an adjusted hazard ratio (HR) of 1.16 (95% CI: 1.13 to 1.19). Among people with a prior AUD registration, alcohol availability was not significantly associated with recurrence of AUD, with an adjusted odds ratio of 1.02 (95% CI: 1.00 to 1.05). Associations of alcohol availability with AUD onset varied according to sex, age, education, neighborhood deprivation, and urbanicity. HRs from the sibling models were similar to those in the general population models, with an adjusted HR = 1.19 (95% CI: 1.15 to 1.24). CONCLUSIONS: Effects varied among neighborhood residents, but greater alcohol availability was a risk factor for AUD onset (but not relapse) in all groups examined except women. Cosibling models suggest there may be a causal relationship of greater alcohol availability with adult-onset AUD.

Platform construction of molecular breeding for utilization of brown macroalgae.

Brown macroalgae are characterized by a large size and high productivity without requiring arable land, fresh water, and fertilizer. Furthermore, since brown macroalgae contain little or no lignin, simple biorefinery processing can efficiently produce sugars from this material. Therefore, brown macroalgae have attracted attention as an alternative feedstock for bioethanol production. However, the utilization of biotechnologies previously developed for terrestrial biomass processing results in difficulties in the bioconversion of brown macroalgae. Recently, several studies have developed biotechnologies for using major carbohydrates of brown macroalgae, such as laminarin, mannitol, and alginate. This review focuses on these fermentation biotechnologies using natural or engineered microorganisms.

Subnational mobility and consumption-based environmental accounting of US corn in animal protein and ethanol supply chains.

Corn production, and its associated inputs, is a relatively large source of greenhouse gas emissions and uses significant amounts of water and land, thus contributing to climate change, fossil fuel depletion, local air pollutants, and local water scarcity. As large consumers of this corn, corporations in the ethanol and animal protein industries are increasingly assessing and reporting sustainability impacts across their supply chains to identify, prioritize, and communicate sustainability risks and opportunities material to their operations. In doing so, many have discovered that the direct impacts of their owned operations are dwarfed by those upstream in the supply chain, requiring transparency and knowledge about environmental impacts along the supply chains. Life cycle assessments (LCAs) have been used to identify hotspots of environmental impacts at national levels, yet these provide little subnational information necessary for guiding firms' specific supply networks. In this paper, our Food System Supply-Chain Sustainability (FoodS3) model connects spatial, firm-specific demand of corn purchasers with upstream corn production in the United States through a cost minimization transport model. This provides a means to link county-level corn production in the United States to firm-specific demand locations associated with downstream processing facilities. Our model substantially improves current LCA assessment efforts that are confined to broad national or state level impacts. In drilling down to subnational levels of environmental impacts that occur over heterogeneous areas and aggregating these landscape impacts by specific supply networks, targeted opportunities for improvements to the sustainability performance of supply chains are identified.

Improvement in ethanol productivity of engineered E. coli strain SSY13 in defined medium via adaptive evolution.

E. coli has the ability to ferment both C5 and C6 sugars and produce mixture of acids along with small amount of ethanol. In our previous study, we reported the construction of an ethanologenic E. coli strain by modulating flux through the endogenous pathways. In the current study, we made further changes in the strain to make the overall process industry friendly; the changes being (1) removal of plasmid, (2) use of low-cost defined medium, and (3) improvement in consumption rate of both C5 and C6 sugars. We first constructed a plasmid-free strain SSY13 and passaged it on AM1-xylose minimal medium plate for 150 days. Further passaging was done for 56 days in liquid AM1 medium containing either glucose or xylose on alternate days. We observed an increase in specific growth rate and carbon utilization rate with increase in passage numbers until 42 days for both glucose and xylose. The 42nd day passaged strain SSK42 fermented 113 g/L xylose in AM1 minimal medium and produced 51.1 g/L ethanol in 72 h at 89% of maximum theoretical yield with ethanol productivity of 1.4 g/L/h during 24-48 h of fermentation. The ethanol titer, yield and productivity were 49, 40 and 36% higher, respectively, for SSK42 as compared to unevolved SSY13 strain.

Fed-batch hydrolysate addition and cell separation by settling in high cell density lignocellulosic ethanol fermentations on AFEX™ corn stover in the Rapid Bioconversion with Integrated recycling Technology process.

The Rapid Bioconversion with Integrated recycling Technology (RaBIT) process uses enzyme and yeast recycling to improve cellulosic ethanol production economics. The previous versions of the RaBIT process exhibited decreased xylose consumption using cell recycle for a variety of different micro-organisms. Process changes were tested in an attempt to eliminate the xylose consumption decrease. Three different RaBIT process changes were evaluated in this work including (1) shortening the fermentation time, (2) fed-batch hydrolysate addition, and (3) selective cell recycling using a settling method. Shorting the RaBIT fermentation process to 11 h and introducing fed-batch hydrolysate addition eliminated any xylose consumption decrease over ten fermentation cycles; otherwise, decreased xylose consumption was apparent by the third cell recycle event. However, partial removal of yeast cells during recycle was not economical when compared to recycling all yeast cells.

Metabolic pathway analysis of the xylose-metabolizing yeast protoplast fusant ZLYRHZ7.

Xylose is the second major fermentable sugar present in hard woods and herbs (after d-glucose). Therefore, efficient conversion of xylose to ethanol is essential for the commercialization of lignocellulosic ethanol, which may provide an ideal alternative to fossil fuels in the future. ZLYRHZ7 is a fusant produced by protoplast fusion between two different yeast species, Saccharomyces cerevisiae W5 and Candida shehatae 20335, which is able to utilize xylose to produce ethanol. To improve ethanol production and to quantitatively analyze metabolic pathway in ZLYRHZ7, we used high performance liquid chromatography (HPLC) to assess the utilization rates of xylose, xylitol, and xylulose, and to measure ethanol yields using xylose, xylitol, and xylulose as sole carbon sources. The ethanol yields reached 0.549±0.003, 0.567±0.003 and 0.544±0.005 g/g in 72 h, which indicated that the metabolic pathways from xylose to xylitol, xylitol to xylulose, and xylulose to ethanol, respectively, were functional. In addition, enzyme activity and qRT-PCR analyses showed that the xylose metabolism-related enzymes xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XK) and their respective genes were expressed at significantly higher levels in ZLYRHZ7 than in both S. cerevisiae W5 and C. shehatae 20335 at 24, 48, and 72 h of fermentation. These results clearly show that the fusant ZLYRHZ7, obtained by protoplast fusion of two different yeast species, has the ability to ferment xylose to produce ethanol.

Bioprospecting thermotolerant ethanologenic yeasts for simultaneous saccharification and fermentation from diverse environments.

Lignocellulosic biomass, a promising renewable energy source, can be used for the production of second generation bioethanol. Simultaneous saccharification and fermentation (SSF), the process which alleviates the problem of separate hydrolysis and fermentation (SHF), requires thermotolerant ethanologenic yeast for bioethanol production. Therefore, ten yeast strains isolated from diverse sources, belonging to various genera like Saccharomyces, Candida, Pichia and Wickerhamomyces were evaluated for their thermotolerance, sugar utilization pattern, inhibitor tolerance and ethanol production potential with glucose, xylose and alkali pretreated paddy straw. All the tested strains were found to be thermotolerant, capable of significant growth at 40°C. Candida tropicalis Y6 was capable of utilizing a wide range of sugars as compared with other yeast isolates. Strains of Candida showed better inhibitor tolerance as compared to Saccharomyces and Pichia strains and exhibited only 5.1-18.8% and 4.7-7.9% reduction in growth with furfural and 5-hydroxymethyl furfural, respectively. Saccharomyces cerevisiae JRC6, isolated from distillery waste, produced ethanol with 88.3% and 89.1% theoretical efficiency at 40°C and 42°C, respectively, from glucose. This strain also produced significantly higher amount of ethanol (3.8 g/L) with better fermentation efficiency (87.9%) from alkali pretreated paddy straw at 40°C, as compared with the other yeast strains. Therefore, S. cerevisiae JRC6, based on its ability to ferment sugars at a higher temperature, can be a promising candidate for production of ethanol from lignocellulosic biomass via SSF process.

Functional expression of xylose isomerase in flocculating industrial Saccharomyces cerevisiae strain for bioethanol production.

Saccharomyces cerevisiae strains with xylose isomerase (XI) pathway were constructed using a flocculating industrial strain (YC-8) as the host. Both strains expressing wild-type xylA (coding XI) from the fungus Orpinomyces sp. and the bacterium Prevotella ruminicola, respectively, showed better growth ability and fermentation capacity when using xylose as the sole sugar than most of the reported strains expressing XI. Codon optimization of both XIs did not improve the xylose fermentation ability of the strains. Adaption significantly increased XI activity resulting in improved growth and fermentation. The strains expressing codon-optimized XI showed a higher increase in xylose consumption and ethanol production compared to strains expressing wild XI. Among all strains, the adapted strain YCPA2E expressing XI from P. ruminicola showed the best performance in the fermentation of xylose to ethanol. After 48 h of fermentation, YCPA2E assimilated 16.95 g/L xylose and produced 6.98 g/L ethanol. These results indicate that YC-8 is a suitable host strain for XI expression, especially for the codon-optimized XI originating from P. ruminicola.

Gender, intoxication and the developing brain: Problematisations of drinking among young adults in Australian alcohol policy.

In this article, we draw on recent scholarly work in the poststructuralist analysis of policy to consider how policy itself functions as a key site in the constitution of alcohol 'problems', and the political implications of these problematisations. We do this by examining Australian alcohol policy as it relates to young adults (18-24 years old). Our critical analysis focuses on three national alcohol policies (1990, 2001 and 2006) and two Victorian state alcohol policies (2008 and 2013), which together span a 25-year period. We argue that Australian alcohol policies have conspicuously ignored young adult men, despite their ongoing over-representation in the statistical 'evidence base' on alcohol-related harm, while increasingly problematising alcohol consumption amongst other population subgroups. We also identify the development of a new problem representation in Australian alcohol policy, that of 'intoxication' as the leading cause of alcohol-related harm and rising hospital admissions, and argue that changes in the classification and diagnosis of intoxication may have contributed to its prioritisation and problematisation in alcohol policy at the expense of other forms of harm. Finally, we draw attention to how preliminary and inconclusive research on the purported association between binge drinking and brain development in those under 25 years old has been mobilised prematurely to support calls to increase the legal purchasing age from 18 to 21 years. Our critical analysis of the treatment of these three issues - gender, intoxication, and brain development - is intended to highlight the ways in which policy functions as a key site in the constitution of alcohol 'problems'.

Increased ethanol production by deletion of HAP4 in recombinant xylose-assimilating Saccharomyces cerevisiae.

The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.

Microbial conversion of pyrolytic products to biofuels: a novel and sustainable approach toward second-generation biofuels.

This review highlights the potential of the pyrolysis-based biofuels production, bio-ethanol in particular, and lipid in general as an alternative and sustainable solution for the rising environmental concerns and rapidly depleting natural fuel resources. Levoglucosan (1,6-anhydrous-ß-D-glucopyranose) is the major anhydrosugar compound resulting from the degradation of cellulose during the fast pyrolysis process of biomass and thus the most attractive fermentation substrate in the bio-oil. The challenges for pyrolysis-based biorefineries are the inefficient detoxification strategies, and the lack of naturally available efficient and suitable fermentation organisms that could ferment the levoglucosan directly into bio-ethanol. In case of indirect fermentation, acid hydrolysis is used to convert levoglucosan into glucose and subsequently to ethanol and lipids via fermentation biocatalysts, however the presence of fermentation inhibitors poses a big hurdle to successful fermentation relative to pure glucose. Among the detoxification strategies studied so far, over-liming, extraction with solvents like (n-butanol, ethyl acetate), and activated carbon seem very promising, but still further research is required for the optimization of existing detoxification strategies as well as developing new ones. In order to make the pyrolysis-based biofuel production a more efficient as well as cost-effective process, direct fermentation of pyrolysis oil-associated fermentable sugars, especially levoglucosan is highlly desirable. This can be achieved either by expanding the search to identify naturally available direct levoglusoan utilizers or modify the existing fermentation biocatalysts (yeasts and bacteria) with direct levoglucosan pathway coupled with tolerance engineering could significantly improve the overall performance of these microorganisms.

Engineering the robustness of Saccharomyces cerevisiae by introducing bifunctional glutathione synthase gene.

Robust, high-yielding Saccharomyces cerevisiae is highly desirable for cost-effective cellulosic ethanol production. In this study, the bifunctional glutathione (GSH) synthetase genes GCSGS at high copy number was integrated into ribosomal DNA of S. cerevisiae by Cre-LoxP system. Threefold higher GSH contents (54.9 µmol/g dry weight) accumulated in the engineered strain BY-G compared to the reference strain. Tolerance of BY-G to H2O2 (3 mM), temperature (40 °C), furfural (10 mM), hydroxymethylfurfural (HMF, 10 mM) and 0.5 mM Cd(2+) increased compared to reference strain. Twofold higher ethanol concentration was obtained by BY-G in simultaneous saccharification and fermentation of corn stover compared to the reference strain. The results showed that intracellular GSH content of S. cerevisiae has an influence on robustness. The strategy is used to engineer S. cerevisiae strains adaptive to a combination of tolerance to inhibitors and raised temperature that may occur in high solid simultaneous saccharification and fermentation of lignocellulosic feedstocks.

Molecular cloning and expression of thermostable glucose-tolerant ß-glucosidase of Penicillium funiculosum NCL1 in Pichia pastoris and its characterization.

A partial peptide sequence of ß-glucosidase isoform (Bgl4) of Penicillium funiculosum NCL1 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cDNA (bgl4) encoding Bgl4 protein was cloned from P. funiculosum NCL1 RNA by consensus RT-PCR. The bgl4 gene encoded 857 amino acids that contained catalytic domains specific for glycoside hydrolase family 3. The cDNA was over-expressed in Pichia pastoris KM71H and the recombinant protein (rBgl4) was purified with the specific activity of 1,354.3 U/mg. The rBgl4 was a glycoprotein with the molecular weight of ~130 kDa and showed optimal activity at pH 5.0 and 60 °C. The enzyme was thermo-tolerant up to 60 °C for 60 min. The rBgl4 was highly active on aryl substrates with ß-glucosidic, ß-xylosidic linkages and moderately active on cellobiose and salicin. It showed remarkably high substrate conversion rate of 3,332 and 2,083 µmol/min/mg with the substrates p-nitrophenyl ß-glucoside and cellobiose respectively. In addition, the rBgl4 showed tolerance to glucose concentration up to 400 mM. It exhibited twofold increase in glucose yield when supplemented with crude cellulase of Trichoderma reesei Rut-C30 in cellulose hydrolysis. These results suggested that rBgl4 is a thermo- and glucose-tolerant ß-glucosidase and is a potential supplement for commercial cellulase in cellulose hydrolysis and thereby assures profitability in bioethanol production.

Bioethanol production from the dry powder of Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae in simultaneous saccharification and fermentation.

It has been found that recombinant Saccharomyces cerevisiae 6525 can produce high concentration of ethanol in one-step fermentation from the extract of Jerusalem artichoke tubers or inulin. However, the utilization rate of raw materials was low and the fermentation process was costly and complicated. Therefore, in this study, after the optimum processing conditions for ethanol production in fed-batch fermentation were determined in flask, the recombinant S. cerevisiae 6525 was first used to produce ethanol from the dry powder of Jerusalem artichoke tubers in 5-L agitating fermentor. After 72 h of fermentation, around 84.3 g/L ethanol was produced in the fermentation liquids, and the conversion efficiency of inulin-type sugars to ethanol was 0.453, or 88.6 % of the theoretical value of 0.511. This study showed high feasibility of bioethanol industrial production from the Jerusalem artichoke tubers and provided a basis for it in the future.