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Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran.

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C6H8O3 and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.

Adaptation, Physiological↗

Quantitative comparison of the signals of an electrochemical bioactivity sensor during the cultivation of different microorganisms.

The microbial activity of different microorganisms was determined by means of an electrochemical bioactivity sensor (BAS). The BAS is based on a biofuel cell and was used for analytical purposes. Online determination of microbial activity using the BAS demonstrated that when different microorganisms with different metabolic pathways were cultivated, a distinct activity signal was detectable with all organisms applied. Furthermore, the results permitted a quantitative comparison of the BAS signals. Among other findings it was shown that the quotient of the BAS signal and the utilized glucose varied from 0.16-29.08 mV g(-1), the quotient of the maximum BAS signal and the released energy of the reaction exhibited a lower variation of 0.07-0.19 mV kJ(-1). Furthermore it was demonstrated that the highest BAS signals could be measured during anaerobic E. coli fermentations, the reason being the formation of electroactive fermentation products, such as formic acid and H(2).

Bacteria, Aerobic↗

Human exposure to oxides of nitrogen at ambient and supra-ambient concentrations.

Oxides of nitrogen (NOx) are a ubiquitous group of air pollutants found in outdoor air as well as indoor environments. The main source of these contaminants is from the combustion of biofuel. Low level and chronic exposure occurs mainly in indoor environments. Acute and high level exposure can occur in a variety of industrial, agricultural, mining, and military settings. The adverse effect of chronic and low level exposure on lung function has been suggested by several epidemiologic studies. However, the results of controlled human exposure to ambient concentrations of NOx have been inconsistent. On the other hand, acute exposure to high levels of NOx has a relatively predictable clinical response in the form of airway irritation, development of pulmonary edema and, in some cases chronic airway disease. There are several lines of evidence to support the role of nutritional antioxidants in amelioration of oxidant lung damage induced by NOx.

Air Pollution↗

Direct electron transfer of glucose oxidase promoted by carbon nanotubes.

A stable suspension of carbon nanotubes (CNT) was obtained by dispersing the CNT in a solution of surfactant, such as cetyltrimethylammonium bromide (CTAB, a cationic surfactant). CNT (dispersed in the solution of 0.1% CTAB) has promotion effects on the direct electron transfer of glucose oxidase (GOx), which was immobilized onto the surface of CNT. The direct electron transfer rate of GOx was greatly enhanced after it was immobilized onto the surface of CNT. Cyclic voltammetric results showed a pair of well-defined redox peaks, which corresponded to the direct electron transfer of GOx, with a midpoint potential of about -0.466 V (vs SCE (saturated calomel electrode)) in the phosphate buffer solution (PBS, pH 6.9). The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (ks) and the value of midpoint potential (E1/2) were estimated. The dependence of E1/2 on solution pH indicated that the direct electron transfer reaction of GOx is a two-electron-transfer coupled with a two-proton-transfer reaction process. The experimental results also demonstrated that the immobilized GOx retained its bioelectrocatalytic activity for the oxidation of glucose, suggesting that the electrode may find use in biosensors (for example, it may be used as a bioanode in biofuel cells). The method presented here can be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.

Aspergillus niger↗

Deactivation of bilirubin oxidase by a product of the reaction of urate and O2.

The "wired" bilirubin oxidase (BOD) bioelectrocatalyst is superior to pure platinum as an electrocatalyst of the four-electron electroreduction of O(2) to water. Not only is its overpotential for O(2) reduction lower, but unlike platinum, it is not affected by organic compounds like glucose. The "wired" BOD-coated carbon cathode operates for >1 week at 37 degrees C in a glucose-containing physiological buffer solution. One of its key applications would be in a glucose-O(2) biofuel cell, which would operate in living tissues. The cathode is, however, short-lived in serum, losing its electrocatalytic activity in a few hours. Here we show that the damaging serum component is a product of the reaction of urate and dissolved oxygen. Exclusion of urate, by application of Nafion film on the cathode, improves the stability in serum.

Catalysis↗

On the stability of the "wired" bilirubin oxidase oxygen cathode in serum.

Oxygen is electroreduced to water on the "wired" bilirubin oxidase (w-BOD) catalyst at a considerably lesser potential than on pure platinum. The w-BOD catalyst could be of value in an implantable glucose-O2 biofuel cell, operating living tissue, if it were stable in serum. We found, however, that w-BOD loses its activity in a few hours in the combined presence of the urate and O2, both of which are normal serum constituents (Bioelectrochemistry, 2004, 65, 83-88). Here we report a second major instability: When the disconnected w-BOD cathode is allowed, in the absence of urate, to poise itself at the potential of the O2/H2O half cell at pH 7.2, it loses its activity rapidly. Unlike the urate/O2 caused loss, this loss can be avoided either by applying a potential that is reducing relative to the O2/H2O half-cell potential, or by excluding O2 and adding a mildly reducing reagent, such as urate. The w-BOD cathode can be stored, therefore, in deoxygenated serum, which contains urate.

Animals↗

Qualitative analysis of products formed during the acid catalyzed liquefaction of bagasse in ethylene glycol.

Bagasse was liquefied in ethylene glycol (EG) catalyzed by sulfuric acid at 190 degrees C under atmospheric pressure. The compositions of the crude products obtained were analyzed after separating them into three fractions: a water-soluble fraction, an acetone-soluble fraction and a residue. With infrared, gel permeation chromatography and elemental analyses, the residue mainly included undissolved cellulose and lignin derivatives and the acetone-soluble fraction mainly contained lignin degradation products with high molecular weights. The water-soluble fraction, after further analyzed by GC-MS and HPLC, showed EG, diethylene glycol, EG derivatives, saccharides, alcohols, aldehydes, ketones, phenols, especially some acids such as formic acid, levulinic acid, acetic acid, oxalic acid and 2-hydroxy-butyric acid and their esters. The Higher Heating Value (HHV) of the residue and the acetone-soluble fractions were higher than that of bagasse. The results showed that some useful chemicals and biofuels could be obtained by this process.

Cellulose↗

Fermentability of eastern gamagrass, big bluestem and sand bluestem grown across a wide variety of environments.

Plant biomass has attracted interest as a feedstock for biofuels production, but much of this work has been focused on relatively few plant species. In this study, three relatively-unstudied species of warm-season perennial grasses, grown at multiple locations in the eastern and central US and harvested over a three year period, were examined for fermentability via in vitro ruminal gas production and dry matter digestibility assays, and near-infrared reflectance calibrations were developed for these fermentation parameters. Big bluestem (Andropogon gerardii Vitman) displayed greater fermentability than did sand bluestem (Andropogon hallii Hack) or eastern gamagrass [Tripsacum dactyloides (L.) L.], but displayed lower biomass yields. The bluestems also displayed lower N contents and less variation in fermentability over different growth environments (geographic locations and harvest years), suggesting a more consistent biomass quality than for eastern gamagrass. Thus, in addition to their use as forage for ruminant animals, bluestems may be of particular interest as feedstocks for bioconversion to ethanol and other products via direct microbial fermentation (consolidated bioprocessing) schemes, and thus merit additional efforts to enhance biomass yield potential.

Agriculture↗

Integrated nanoparticle-biomolecule systems for biosensing and bioelectronics.

The similar dimensions of biomolecules such as enzymes, antibodies or DNA, and metallic or semiconductor nanoparticles (NPs) enable the synthesis of biomolecule-NP hybrid systems where the unique electronic, photonic and catalytic properties of NPs are combined with the specific recognition and biocatalytic properties of biomolecules. The unique functions of biomolecule-NP hybrid systems are discussed with several examples: (i) the electrical contacting of redox enzymes with electrodes is the basis for the development of enzymatic electrodes for amperometric biosensors or biofuel cell elements. The reconstitution of the apo-glucose oxidase or apo-glucose dehydrogenase on flavin adenine dinucleotide (FAD)-functionalized Au NPs (1.4 nm) associated with electrodes, or on pyrroloquinoline quinone (PQQ)-functionalized Au NPs (1.4 nm) associated with electrodes, respectively, yields electrically contacted enzyme electrodes. The aligned, reconstituted enzymes on the electrode surfaces reveal effective electrical contacting, and the glucose oxidase and glucose dehydrogenase reveal turnover rates of 5000 and 11,800 s(-1), respectively. (ii) The photoexcitation of semiconductor nanoparticles yields fluorescence with a wavelength controlled by the size of the NPs. The fluorescence functions of semiconductor NPs are used to develop a fluorescence resonance energy transfer (FRET) assay for nucleic acids, and specifically, for analyzing telomerase activity in cancer cells. CdSe-ZnS NPs are functionalized by a primer recognized by telomerase, and this is elongated by telomerase extracted from HeLa cancer cells in the presence of dNTPs and Texas-red-functionalized dUTP. The dye integrated into the telomers allows the FRET process that is intensified as telomerization proceeds. Also, the photoexcited electron-hole pair generated in semiconductor NPs is used to generate photocurrents in a CdS-DNA hybrid system associated with an electrode. A redox-active intercalator, methylene blue, was incorporated into a CdS-duplex DNA monolayer associated with a Au electrode, and this facilitated the electron transfer between the electrode and the CdS NPs. The direction of the photocurrent was controlled by the oxidation state of the intercalator. (iii) Biocatalysts grow metallic NPs, and the absorbance of the NPs provides a means to assay the biocatalytic transformations. This is exemplified with the glucose oxidase-induced growth of Au NPs and with the tyrosinase-stimulated growth of Au NPs, in the presence of glucose or tyrosine, respectively. The biocatalytic growth of the metallic NPs is used to grow nanowires on surfaces. Glucose oxidase or alkaline phosphatase functionalized with Au NPs (1.4 nm) acted as 'biocatalytic inks' for the synthesis of metallic nanowires. The deposition of the Au NP-modified glucose oxidase, or the Au NP-modified alkaline phosphatase on Si surfaces by dip-pen nanolithography led to biocatalytic templates, that after interaction with glucose/AuCl4- or p-aminophenolphosphate/Ag+, allowed the synthesis of Au nanowires or Ag nanowires, respectively.

Biosensing Techniques↗

Direct electron transfer with yeast cells and construction of a mediatorless microbial fuel cell.

The direct electron transfer exhibited by the yeast cells, Hansenula anomala has been demonstrated using the electrochemical technique cyclic voltammetry by immobilizing the microorganisms by two different methods viz., physical adsorption and covalent linkage. The analysis of redox enzymes present in the outer membrane of the microorganisms has been carried out in this work. This paper demonstrates that yeast cells with redox enzymes present in their outer membrane are capable of communicating directly with the electrode surface and contribute to current generation in a mediatorless biofuel cells. The efficiency of current generation has been evaluated using three anode materials.

Bioelectric Energy Sources↗

Electron-conducting redox hydrogels: Design, characteristics and synthesis.

Redox hydrogels constitute the only electron-conducting phase in which water-soluble chemicals and biochemicals dissolve and diffuse. The combination of solubility and diffusion makes the electron-conducting gels permeable to water-soluble biochemicals and chemicals. The electron-conducting redox hydrogels serve to electrically connect the redox centers of enzymes to electrodes, enabling their use whenever leaching of electron-shuttling diffusional redox mediators must be avoided, which is the case in subcutaneously implanted biosensors for diabetes management and in miniature, potentially implantable, glucose-O2 biofuel cells. Because the hydrogels envelope the redox enzymes, they electrically wire the reaction centers to electrodes irrespective of spatial orientation and connect to electrode redox centers of multiple enzyme layers. Hence, the attained current densities of enzyme substrate electrooxidation or electroreduction are much higher than with enzyme monolayers packed onto electrode surfaces.

Electrons↗

Protein engineering in bioelectrocatalysis.

Electrochemistry of redox proteins is a broadly applicable technology with important applications in biosensors, biofuel cells and chemical syntheses. Escalating attention in this area is driven by remarkable progress in designing efficient interfaces for transferring electrons between electrode surfaces and redox proteins. Research in interface design is slowly shifting from modifying electrode surfaces towards the engineering of redox proteins. Protein engineering, which encompasses rational design, directed evolution and combined methods, offers many powerful methods and strategies for improving the electron transfer properties of redox proteins.

Catalysis↗

Electrical contacting of redox proteins by nanotechnological means.

Redox enzymes in bioelectronic devices usually lack direct electrical contact with electrodes, owing to the spatial separation of their redox centers from the conductive surfaces by the protein shells. The reconstitution of apo-enzymes on cofactor-functionalized nanostructures associated with electrodes provides a means to align the biocatalysts on the conductive surface and to electrically contact redox enzymes with electrodes. The reconstitution of apo-enzymes on cofactor-functionalized gold nanoparticles or carbon nanotubes has led to effective electrical communication between the redox proteins and the electrodes. Alternatively, the reconstitution of redox enzymes on molecular wires that enable electron tunneling or dynamic charge shuttling represent supramolecular biocatalytic nanostructures exhibiting electrical contact. The bioelectrocatalytic activities of the electrically wired reconstituted enzymes on electrodes have allowed the development of amperometric biosensors and biofuel cell elements.

Biosensing Techniques↗

The setting of standards for agricultural nitrogen emissions: a case study of the Delphi technique.

The Delphi technique is a means of aggregating the judgement of a panel of experts in order to improve the quality of decision-making. This paper provides a case study of the technique by undertaking a three-round Delphi study to determine a package of best available techniques to reduce nitrogen emissions from a poultry unit under the Integrated Pollution Prevention and Control Directive (IPPC). Forms of nitrogen addressed included ammonia (NH3), nitrous oxide (N2O) nitrogen oxides (NOx), dusts and nitrate (NO3-), with the study providing a means to prioritise the pollution concerns on different spatial scales. The priority pollutant issues were the contribution of NH3 to eutrophication, the global cooling effect of NH4+ aerosol, the role of NH4+ as a vector for atmospheric transport of NOx and SO2, the contribution of N2O to global warming, and NO3- leaching. Reduced nitrogen (NHx) was rated as a priority on all scales, while N2O and NO3- were rated as priorities only on global and local scales, respectively. The study indicated the need for abatement techniques at each stage of poultry rearing and waste management, with particular attention to reduce NH3 emissions, reflecting the priority pollutant concerns. Measures identified by the panel include maintenance of dry litter, low emission removal of litter from housing and storage of litter under cover. Once the litter has left the farm, this should either be used as a biofuel for electricity generation or rapidly incorporated into agricultural soils. The amounts and timing of manure application should be tuned to crop needs. Uncertainties in the Delphi technique limit its suitability as a stand-alone decision making tool. However, the Delphi technique proved useful in identifying priority pollutant issues, areas of agreement, disagreement and where information is lacking. This demonstrates its use when dealing with the complex issues of prioritising pollution issues and abatement approaches.

Air Pollutants↗

Prospects for cleaning ash in the acidic effluent from bioleaching of sulfidic concentrates.

Leaching of ashes in sulfuric acid (pH 1.0, liquid-to-solid (L/S) ratio 10:1, 25 degrees C) has been characterized with respect to the neutralizing capacity and the dissolution of dominant ions and trace elements. The conditions mimic the oxidation stage of a biohydrometallurgical process for base metal production from sulfidic mineral concentrates. Direct acid leaching of ash, integrated with this metallurgical process, offers a feasible route to the sustainable handling of metal-rich ashes. The treated ash will be deposited together with the inert mineral residue. Cd, Co, Cu, Ni and Zn are effectively leached and can be recovered utilizing existing hydrometallurgical technology, but the recovery of other readily dissolved metals, notably Mn, U and V, requires that additional steps are implemented. We make two recommendations for industrial processes. The first is to replace limestone with ash from biofuels, except peat, for pH control in biohydrometallurgical processing. This requires a modest increase of fresh alkali compared with limestone. The second is to implement sulfuric acid leaching of fly ash from the combustion of solid waste and other metal-rich fuels (used wood, tires), thereby avoiding costly ash-deposits. There is a significant economic incentive for these changes, since no costly ash-deposits and less limestone will be needed.

Hazardous Waste↗

Effects on growth, photosynthesis and pigments of the freshwater moss Fontinalis antipyretica Hedw. after exposure to wood ash solution.

The application of wood ash from biofuel sources to the forest has been suggested as a source of nutrients for trees and for restoration of acidified soils and surface waters. Studies on the effects of wood ash on aquatic organisms and ecosystems are, however, few. This study investigated the effects of wood ash (0.1-10 g l(-1)) on the freshwater moss Fontinalis antipyretica Hedw., which has previously been shown to be a sensitive test organism for assessing environmental pollution. After nine weeks of treatment with wood ash, a significant effect of enhanced stem growth was observed at higher concentrations (1-10 g l(-1)). The concentration of wood ash was also correlated with the growth of secondary branches. Photosynthesis (oxygen evolution after 4 h of exposure to wood ash) was significantly lower in the alkaline treatments (no pH adjustment) compared to the treatments with neutral wood ash solutions (pH adjusted to 7.5). Furthermore, photosynthesis in the alkaline wood ash treatments was significantly lower compared to the control. The ratio between the optical density value before and after acidification (OD(665/665a)) was higher for all wood ash concentrations compared to the control. The OD(665/665a) values ranged from 1.52 to 1.61 and there was a difference, however not significant, between the alkaline and the neutral treatment at 10 g l(-1) wood ash. This study clearly demonstrated that wood ash may be beneficial for F. antipyretica at moderate concentrations (0.5-5 g l(-1)), particularly when a sudden increase in pH is avoided.

Bryopsida↗

Crop biotechnology provides an opportunity to develop a sustainable future.

The current reliance on petro-based fuels and chemicals is not sustainable. New technologies typically take approximately 25 years to penetrate the market; consequently, the development of viable alternatives is required in the near future. Plant-based systems capture solar energy and can be produced in a renewable manner. However, the harvestable parts are not well optimized for energy transfer and this has been a significant limitation to the development of economically viable and sustainable biomass energy systems. Biotechnology has provided a new toolset that can be used to design and optimize the capture of solar energy through crops. Further development of biotechnology and genomics tools will enable the development of crops with specific traits that are optimized for biofuels and bioenergy. The implementation of such a system will enable a sustainable platform for centuries to come and should be given a high priority in society.

Agriculture↗

PCDD/F in source-sorted waste fractions and emissions from their co-combustion with reed canary-grass.

The dry combustible fraction of source-sorted household waste, including material that would otherwise be recycled, was mixed with the energy crop reed canary-grass (Phalaris arundinacea L.), and combusted as briquettes in 150 and 600 kW biofuel-boilers without advanced cleaning systems. The source-sorted waste was further sorted and characterized according to its material and chemical contents. The bulk of the waste's chlorine content came from the non-package plastic fraction, whereas 90-95% of summation operator PCDD/F (74-90% of WHO-TEQ) originated from the textile fraction. The sources of the dioxins in the waste fractions are discussed. The balance of dioxin levels was negative, i.e., the amounts of dioxins output in the flue gas were lower than those input in the fuel, except when there were operational disturbances in the combustion. In one of the combustion trials the total levels of dioxins in the flue-gas and ashes were also lower than the input levels. The use of additional cleaning equipment will be needed to ensure that emissions of dioxins and hydrochloric acid will be below legal limits.

Biodegradation, Environmental↗