Ethanol Fuels: E10 or E85 – Life Cycle Perspectives (5 pp)

Goal and Scope  

The environmental performance of two ethanol fuel applications (E10 and E85) is compared (E10 fuel: a mixture of 10% ethanol and 90% gasoline by volume, and E85 fuel: a mixture of 85% ethanol and 15% gasoline by volume).     

Quantifying the metabolic capabilities of engineered <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> using linear programming analysis

Background  

The need for discovery of alternative, renewable, environmentally friendly energy sources and the development of cost-efficient, "clean" methods for their conversion into higher fuels becomes imperative. Ethanol, whose significance as fuel has dramatically increased in the last decade, can be produced from hexoses and pentoses through microbial fermentation. Importantly, plant biomass, if appropriately and effectively decomposed, is a potential inexpensive and highly renewable source of the hexose and pentose mixture. Recently, the engineered (to also catabolize pentoses) anaerobic bacterium Zymomonas mobilis has been widely discussed among the most promising microorganisms for the microbial production of ethanol fuel. However, Z. mobilis genome having been fully sequenced in 2005, there is still a small number of published studies of its in vivo physiology and limited use of the metabolic engineering experimental and computational toolboxes to understand its metabolic pathway interconnectivity and regulation towards the optimization of its hexose and pentose fermentation into ethanol.     

Life cycle assessment of switchgrass-derived ethanol as transport fuel

Background, aim, and scope  

The increasing gasoline price, the depletion of fossil resources, and the negative environmental consequences of driving with petroleum fuels have driven the development of alternative transport fuels. Bioethanol, which is converted from cellulosic feedstocks, has attracted increasing attention as one such alternative. This study assesses the environmental impact of using ethanol from switchgrass as transport fuel and compares the results with the ones of gasoline to analyze the potential of developing switchgrass ethanol as an environmentally sustainable transport fuel.     

Genetic variation for parental effects on the propensity to gregarise in <Emphasis Type="Italic">Locusta migratoria</Emphasis>

Background  

Environmental parental effects can have important ecological and evolutionary consequences, yet little is known about genetic variation among populations in the plastic responses of offspring phenotypes to parental environmental conditions. This type of variation may lead to rapid phenotypic divergence among populations and facilitate speciation. With respect to density-dependent phenotypic plasticity, locust species (Orthoptera: family Acrididae), exhibit spectacular developmental and behavioural shifts in response to population density, called phase change. Given the significance of phase change in locust outbreaks and control, its triggering processes have been widely investigated. Whereas crowding within the lifetime of both offspring and parents has emerged as a primary causal factor of phase change, less is known about intraspecific genetic variation in the expression of phase change, and in particular in response to the parental environment. We conducted a laboratory experiment that explicitly controlled for the environmental effects of parental rearing density. This design enabled us to compare the parental effects on offspring expression of phase-related traits between two naturally-occurring, genetically distinct populations of Locusta migratoria that differed in their historical patterns of high population density outbreak events.     

Life cycle assessment of fuel ethanol from cane molasses in Thailand

Background, aim and scope

After China and India, Thailand is considered another emerging market for fuel ethanol in Asia. At present, ethanol in the country is mainly a fermentation/distillery product of cane molasses, although cassava and cane juice are considered other potential raw materials for the fuel. This study aims to evaluate the environmental impacts of substituting conventional gasoline (CG) with molasses-based gasohol in Thailand.

Materials and methods

The life cycle assessment (LCA) procedure carried out follows three interrelated phases: inventory analysis, characterization and interpretation. The functional unit for the comparison is 1 l gasoline equivalent consumed by a new passenger car to travel a specific distance.

Results

The results of the study show that molasses-based ethanol (MoE) in the form of 10% blend with gasoline (E10), along its whole life cycle, consumes less fossil energy (5.3%), less petroleum (8.1%) and provides a similar impact on acidification compared to CG. The fuel, however, has inferior performance in other categories (e.g. global warming potential, nutrient enrichment and photochemical ozone creation potential) indicated by increased impacts over CG.

Discussion

In most cases, higher impacts from the upstream of molasses-based ethanol tend to govern its net life cycle impacts relative to CG. This makes the fuel blend less environmentally friendly than CG for the specific conditions considered. However, as discussed later, this situation can be improved by appropriate changes in energy carriers.

Conclusions

The LCA procedure helps identify the key areas in the MoE production cycle where changes are required to improve environmental performance. Specifically, they are: (1) use of coal as energy source for ethanol conversion, (2) discharge of distillery spent wash into an anaerobic pond, and (3) open burning of cane trash in sugar cane production.

Recommendations and perspectives

Measures to improve the overall life cycle energy and environmental impacts of MoE are: (1) substituting biomass for fossil fuels in ethanol conversion, (2) capturing CH₄ from distillery spent wash and using it as an energy supply, and (3) utilizing cane trash for energy instead of open burning in fields.     

Impact of the <Emphasis Type="Italic">rpoS</Emphasis> genotype for acid resistance patterns of pathogenic and probiotic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σ^S, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors.     

Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop‐mediated isothermal amplification (LAMP) reaction

Aims

To develop two assays based on the loop‐mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes.

Methods and Results

Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction.

Conclusions

The two LAMP assays permitted to quickly and specifically identify DNA from OTA‐producing black aspergilli, as well as isolates grown in pure culture.

Significance and Impact of the Study

Monitoring vineyards for the presence of OTA‐producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA‐producing black aspergilli.     

Metabolic regulation analysis of an ethanologenic <Emphasis Type="Italic">Escherichia coli</Emphasis> strain based on RT-PCR and enzymatic activities

Background  

A metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14) derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDC _Zm and ADH _Zm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis). It is suggested that this behavior might be due to lineage differences between E. coli W and C.     

Why does oriental arborvitae grow better when mixed with black locust: Insight on nutrient cycling?

To identify why tree growth differs by afforestation type is a matter of prime concern in forestry. A study was conducted to determine why oriental arborvitae (Platycladus orientalis) grows better in the presence of black locust (Robinia pseudoacacia) than in monoculture. Different types of stands (i.e., monocultures and mixture of black locust and oriental arborvitae, and native grassland as a control) were selected in the Loess Plateau, China. The height and diameter at breast height of each tree species were measured, and soil, shoot, and root samples were sampled. The arbuscular mycorrhizal (AM) attributes, shoot and root nutrient status, height and diameter of black locust were not influenced by the presence of oriental arborvitae. For oriental arborvitae, however, growing in mixture increased height and diameter and reduced shoot Mn, Ca, and Mg contents, AM fungal spore density, and colonization rate. Major changes in soil properties also occurred, primarily in soil water, NO₃‐N, and available K levels and in soil enzyme activity. The increase in soil water, N, and K availability in the presence of black locust stimulated oriental arborvitae growth, and black locust in the mixed stand seems to suppress the development of AM symbiosis in oriental arborvitae roots, especially the production of AM fungal spores and vesicles, through improving soil water and N levels, thus freeing up carbon to fuel plant growth. Overall, the presence of black locust favored oriental arborvitae growth directly by improving soil water and fertility and indirectly by repressing AM symbiosis in oriental arborvitae roots.     

Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD⁺-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys²⁷⁴→Arg Asn²⁷⁶→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.     

Loop‐mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp

Aims

Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop‐mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite.

Methods and Results

A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 min at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA‐labelled nanogold probe, followed by salt‐induced AuNP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0·02 fg total DNA) than a conventional nested PCR (0·2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP‐AuNP assay was specific for E. hepatopenaei.

Conclusions

Without sacrificing sensitivity or specificity, the new LAMP‐AuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp.

Significance and Impact of the study

The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.     

Susceptibility of Pediococcus isolates to antimicrobial compounds in relation to hop-resistance and beer-spoilage

Background  

Though important in the context of food microbiology and as potential pathogens in immuno-compromised humans, bacterial isolates belonging to the genus Pediococcus are best known for their association with contamination of ethanol fermentation processes (beer, wine, or fuel ethanol). Use of antimicrobial compounds (e.g., hop-compounds, Penicillin) by some industries to combat Pediococcus contaminants is long-standing, yet knowledge about the resistance of pediococci to antimicrobial agents is minimal. Here we examined Pediococcus isolates to determine whether antibiotic resistance is associated with resistance to hops, presence of genes known to correlate with beer spoilage, or with ability to grow in beer.     

The genome-scale metabolic network analysis <Emphasis Type="Italic">of Zymomonas mobilis</Emphasis> ZM4 explains physiological features and suggests ethanol and succinic acid production strategies

Background  

Zymomonas mobilis ZM4 is a Gram-negative bacterium that can efficiently produce ethanol from various carbon substrates, including glucose, fructose, and sucrose, via the Entner-Doudoroff pathway. However, systems metabolic engineering is required to further enhance its metabolic performance for industrial application. As an important step towards this goal, the genome-scale metabolic model of Z. mobilis is required to systematically analyze in silico the metabolic characteristics of this bacterium under a wide range of genotypic and environmental conditions.     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.     

Environmental amoebae do not support the long‐term survival of virulent mycobacteria

Aims

To test the hypothesis that Mycobacterium bovis can persist in the environment within protozoa.

Methods and Results

In this study, we used a novel approach to detect internalized mycobacteria in environmental protozoa from badger latrines. Acid‐fast micro‐organisms were visualized in isolated amoebae, although we were unable to identify them to species level as no mycobacteria were grown from these samples nor was M. bovis detected by IS6110 PCR. Co‐incubation of Acanthamoeba castellanii with virulent M. bovis substantially reduced levels of bacilli, indicating that the amoebae have a negative effect on the persistence of M. bovis.

Conclusions

The internalization of mycobacteria in protozoa might be a rare event under environmental conditions. The results suggest that amoebae might contribute to the inactivation of M. bovis rather than representing a potential environmental reservoir.

Significance and Impact of the Study

Protozoa have been suggested to act as an environmental reservoir for M. bovis. The current study suggests that environmental amoebae play at most a minor role as potential reservoirs of M. bovis and that protozoa might inhibit persistence of M. bovis in the environment.     

Nitrogen stocks in a riparian area invaded by N-fixing black locust (Robinia pseudoacacia L.)

Fixation of river flow passages and riverbed degradation may facilitate the development of higher floodplains with the establishment of exotic species such as black locust (Robinia pseudoacacia L.). We quantitatively evaluated the relationship between nitrogen (N) levels in black locust trees and in sediments under different flood disturbance regimes in a riparian area of the Chikuma River. In this study, allometric equations were developed for relating leaf N content to diameter at breast height of black locust. The amount of leaf N in black locust increased with distance from the river, reaching 159 kg N ha⁻¹ at 180 m from the river. There was a small difference in N content between green and fallen leaves (0.2%), and so the leaf N was almost equivalent to N input to riparian sediments. Fine sediments accumulated on the riparian area, where the amount of sediments N increased with distance from the river, ranging from 1091 ± 767 to 4953 ± 2953 kg N ha⁻¹. The N accumulation rates also increased with distance from the river, corresponding with the amount of leaf N in black locust per unit area, but the former exceeded the latter. The sediment N accumulation might be accelerated by sediment trapping effect due to riparian vegetation itself. A large input of N provided by invaded black locust might alter nutrient dynamics and native plant community structure in the riparian area.     

Inhibition of hydrogen uptake in <Emphasis Type="Italic">Escherichia coli</Emphasis> by expressing the hydrogenase from the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis>sp. PCC 6803

Background  

Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H⁺ + 2e^- ↔ H₂ (g).     

Evaluation of Petrifilm™ Select <Emphasis Type="Italic">E. coli</Emphasis> Count Plate medium to discriminate antimicrobial resistant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Screening and enumeration of antimicrobial resistant Escherichia coli directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli.     

A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>, <Emphasis Type="Italic">E. moshkovskii</Emphasis> and <Emphasis Type="Italic">E. dispar</Emphasis> DNA in stool samples

Background  

E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples.     

Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>for biotechnological application

Background  

Plant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system.