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.

Sustainable bioenergy production from marginal lands in the US Midwest.

Legislation on biofuels production in the USA and Europe is directing food crops towards the production of grain-based ethanol, which can have detrimental consequences for soil carbon sequestration, nitrous oxide emissions, nitrate pollution, biodiversity and human health. An alternative is to grow lignocellulosic (cellulosic) crops on 'marginal' lands. Cellulosic feedstocks can have positive environmental outcomes and could make up a substantial proportion of future energy portfolios. However, the availability of marginal lands for cellulosic feedstock production, and the resulting greenhouse gas (GHG) emissions, remains uncertain. Here we evaluate the potential for marginal lands in ten Midwestern US states to produce sizeable amounts of biomass and concurrently mitigate GHG emissions. In a comparative assessment of six alternative cropping systems over 20 years, we found that successional herbaceous vegetation, once well established, has a direct GHG emissions mitigation capacity that rivals that of purpose-grown crops (-851 ± 46 grams of CO(2) equivalent emissions per square metre per year (gCO(2)e m(-2) yr(-1))). If fertilized, these communities have the capacity to produce about 63 ± 5 gigajoules of ethanol energy per hectare per year. By contrast, an adjacent, no-till corn-soybean-wheat rotation produces on average 41 ± 1 gigajoules of biofuel energy per hectare per year and has a net direct mitigation capacity of -397 ± 32 gCO(2)e m(-2) yr(-1); a continuous corn rotation would probably produce about 62 ± 7 gigajoules of biofuel energy per hectare per year, with 13% less mitigation. We also perform quantitative modelling of successional vegetation on marginal lands in the region at a resolution of 0.4 hectares, constrained by the requirement that each modelled location be within 80 kilometres of a potential biorefinery. Our results suggest that such vegetation could produce about 21 gigalitres of ethanol per year from around 11 million hectares, or approximately 25 per cent of the 2022 target for cellulosic biofuel mandated by the US Energy Independence and Security Act of 2007, with no initial carbon debt nor the indirect land-use costs associated with food-based biofuels. Other regional-scale aspects of biofuel sustainability, such as water quality and biodiversity, await future study.

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.

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.

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.

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.

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.

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.

Effects of yeast immobilization on bioethanol production.

The current study evaluated a newer method, which includes a dehydration step, of immobilizing Saccharomyces cerevisiae L-77 and S. cerevisiae L-73 onto hydroxylapatite and chamotte ceramic supports. The efficiency of cell immobilization on chamotte was significantly higher than hydroxylapatite. Immobilized yeast preparations were investigated for their ethanol-producing capabilities. The glucose concentration in a fermentation medium was 100 mg/mL. Immobilized preparations produced the same amount of ethanol (48 ± 0.5 mg/mL) as free cells after 36 H of fermentation. During the early stages of fermentation, immobilized yeast cells produced ethanol at a higher rate than free cells. Yeast preparations immobilized on both supports (hydroxylapatite and chamotte) were successfully used in six sequential batch fermentations without any loss of activity. The chamotte support was more stable in the fermentation medium during these six cycles of ethanol production. In addition to the high level of ethanol produced by cells immobilized on chamotte, the stability of this support and its low cost make it a promising material for biotechnologies associated with ethanol production.

Evaluation of hardboard manufacturing process wastewater as a feedstream for ethanol production.

Waste streams from the wood processing industry can serve as feedstream for ethanol production from biomass residues. Hardboard manufacturing process wastewater (HPW) was evaluated on the basis of monomeric sugar recovery and fermentability as a novel feedstream for ethanol production. Dilute acid hydrolysis, coupled with concentration of the wastewater resulted in a hydrolysate with 66 g/l total fermentable sugars. As xylose accounted for 53 % of the total sugars, native xylose-fermenting yeasts were evaluated for their ability to produce ethanol from the hydrolysate. The strains selected were, in decreasing order by ethanol yields from xylose (Y p/s, based on consumed sugars), Scheffersomyces stipitis ATCC 58785 (CBS 6054), Pachysolen tannophilus ATCC 60393, and Kluyveromyces marxianus ATCC 46537. The yeasts were compared on the basis of substrate utilization and ethanol yield during fermentations of the hydrolysate, measured using an HPLC. S. stipitis, P. tannophilus, and K. marxianus produced 0.34, 0.31, and 0.36 g/g, respectively. The yeasts were able to utilize between 58 and 75 % of the available substrate. S. stipitis outperformed the other yeast during the fermentation of the hydrolysate; consuming the highest concentration of available substrate and producing the highest ethanol concentration in 72 h. Due to its high sugar content and low inhibitor levels after hydrolysis, it was concluded that HPW is a suitable feedstream for ethanol production by S. stipitis.

Mortality of harmful drinkers increased after reduction of alcohol prices in northern Finland: a 10-year follow-up of head trauma subjects.

OBJECTIVE: Alcohol-related mortality may be influenced by the level of alcohol consumption. We investigated the effect of alcohol price reduction on mortality in a cohort of 827 subjects with head injury. METHODS: We used the Finnish National Hospital Discharge Register to identify all diagnoses recorded during hospital and health center visits for survivors of the index injury during a follow-up of 10 years. Mortality data were gathered from death records obtained from the Official Cause-of-Death Statistics. Cox proportional hazards model was used to identify independent predictors for death. Kaplan-Meier survival curves were used to characterize the effect of alcohol price reduction on mortality of harmful and non-harmful drinkers. RESULTS: Alcohol-related deaths increased after the reduction of alcohol prices on March 1, 2004. Subjects recorded as harmful drinkers during the follow-up period were significantly (p < 0.001) more likely than others to die after the price reduction. Older age (HR 1.06, 95% CI 1.05-1.07), moderate-to-severe brain injury (HR 2.39, 95% CI 1.59-3.60) and harmful drinking recorded after the index trauma (HR 2.59, 95% CI 1.62-4.62) were significant (p < 0.001) predictors for death. CONCLUSION: We conclude that a political decision to lower the price of alcohol may cause a significant increase in the death rate of harmful drinkers.

Structural changes of corn stover lignin during acid pretreatment.

In this study, raw corn stover was subjected to dilute acid pretreatments over a range of severities under conditions similar to those identified by the National Renewable Energy Laboratory (NREL) in their techno-economic analysis of biochemical conversion of corn stover to ethanol. The pretreated corn stover then underwent enzymatic hydrolysis with yields above 70 % at moderate enzyme loading conditions. The enzyme exhausted lignin residues were characterized by ³¹P NMR spectroscopy and functional moieties quantified and correlated to enzymatic hydrolysis yields. Results from this study indicated that both xylan solubilization and lignin degradation are important for improving the enzyme accessibility and digestibility of dilute acid pretreated corn stover. At lower pretreatment temperatures, there is a good correlation between xylan solubilization and cellulose accessibility. At higher pretreatment temperatures, lignin degradation correlated better with cellulose accessibility, represented by the increase in phenolic groups. During acid pretreatment, the ratio of syringyl/guaiacyl functional groups also gradually changed from less than 1 to greater than 1 with the increase in pretreatment temperature. This implies that more syringyl units are released from lignin depolymerization of aryl ether linkages than guaiacyl units. The condensed phenolic units are also correlated with the increase in pretreatment temperature up to 180 °C, beyond which point condensation reactions may overtake the hydrolysis of aryl ether linkages as the dominant reactions of lignin, thus leading to decreased cellulose accessibility.

Fungal ß-glucosidase expression in Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae strains expressing ß-glucosidases from Thermoascus aurantiacus (Tabgl1) and Phanerochaete chrysosporium (PcbglB and Pccbgl1) were constructed and compared to S. cerevisiae Y294[SFI], previously identified as the best ß-glucosidase-producing strain. The PcbglB was also intracellularly expressed in combination with the lac12 lactose permease of Kluyveromyces lactis in S. cerevisiae Y294[PcbglB + Lac12]. The recombinant extracellular ß-glucosidases indicated maximum activity in the pH range 4-5 and temperature optima varying from 50 to 75 °C. The S. cerevisiae Y294[Pccbgl1] strain performed best under aerobic and anaerobic conditions, producing 2.6 times more ß-glucosidase activity than S. cerevisiae Y294[SFI] and an ethanol concentration of 4.8 g l(-1) after 24 h of cultivation on cellobiose as sole carbohydrate source. S. cerevisiae Y294[Tabgl1] was unable to grow on cellobiose (liquid medium), whereas S. cerevisiae Y294[PcbglB + Lac12] exhibited limited growth.