The second-generation (2G) fermentation environment for lignocellulose conversion presents unique challenges to the fermentative organism that do not necessarily exist in other industrial ...fermentations. While extreme osmotic, heat, and nutrient starvation stresses are observed in sugar- and starch-based fermentation environments, additional pre-treatment-derived inhibitor stress, potentially exacerbated by stresses such as pH and product tolerance, exist in the 2G environment. Furthermore, in a consolidated bioprocessing (CBP) context, the organism is also challenged to secrete enzymes that may themselves lead to unfolded protein response and other stresses. This review will discuss responses of the yeast
Saccharomyces cerevisiae
to 2G-specific stresses and stress modulation strategies that can be followed to improve yeasts for this application. We also explore published –omics data and discuss relevant rational engineering, reverse engineering, and adaptation strategies, with the view of identifying genes or alleles that will make positive contributions to the overall robustness of 2G industrial strains.
Keypoints
• Stress tolerance is a key driver to successful application of yeast strains in biorefineries.
• A wealth of data regarding stress responses has been gained through omics studies.
• Integration of this knowledge could inform engineering of fit for purpose strains.
Major breakthroughs have recently been reported that can help overcome two inherent drawbacks of NMR: the lack of sensitivity and the limited memory of longitudinal magnetization. Dynamic nuclear ...polarization (DNP) couples nuclear spins to the large reservoir of electrons, thus making it possible to detect dilute endogenous substances in magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). We have designed a method to preserve enhanced ("hyperpolarized") magnetization by conversion into long-lived states (LLS). It is shown that these enhanced long-lived states can be generated for proton spins, which afford sensitive detection. Even in complex molecules such as peptides, long-lived proton states can be sustained effectively over time intervals on the order of tens of seconds, thus allowing hyperpolarized substrates to reach target areas and affording access to slow metabolic pathways. The natural abundance carbon-13 polarization has been enhanced ex situ by almost four orders of magnitude in the dipeptide Ala-Gly. The sample was transferred by the dissolution process to a high-resolution magnet where the carbon-13 polarization was converted into a long-lived state associated with a pair of protons. In Ala-Gly, the lifetime TLLS associated with the two nonequivalent Hα glycine protons, sustained by suitable radio-frequency irradiation, was found to be seven times longer than their spin-lattice relaxation time constant (TLLS/T₁ = 7). At desired intervals, small fractions of the populations of long-lived states were converted into observable magnetization. This opens the way to observing slow chemical reactions and slow transport phenomena such as diffusion by enhanced magnetic resonance.
We present the status of development of a novel neutron spin filter based on the strong spin dependence of the neutron scattering on protons. Using photo-excited triplet states for the dynamic ...nuclear polarization (DNP) process, proton polarization values of 80% can now be achieved in pentacene doped single crystals of naphthalene at a field of 0.36 T in a simple helium flow cryostat. Careful sample preparation lead to extremely long polarization decay times under moderate conditions that allowed us to develop a transportable device. The filter is polarized in the lab under well controlled conditions and is then transferred to the neutron beam line where it can be operated during several days with almost constant polarization while requiring only a minimum of equipment. We describe briefly the main features of the spin filter and its use as a spin analyzer in a small-angle neutron scattering (SANS) experiment probing the magnetic structure of a nanocrystalline soft magnetic material. Furthermore the procedure for performing the background and spin leakage corrections of the neutron data is outlined in detail.
The use of polarized protons as neutron spin filter is an attractive alternative to the well established neutron polarization techniques, as the large, spin-dependent neutron scattering cross-section ...for protons is useful up to the sub-MeV region. Employing optically excited triplet states for the dynamic nuclear polarization (DNP) of the protons relieves the stringent requirements of classical DNP schemes, i.e low temperatures and strong magnetic fields, making technically simpler systems with open geometries possible.
Using triplet DNP a record polarization of 71% has been achieved in a pentacene doped naphthalene single crystal at a field of 0.36T using a simple helium flow cryostat for cooling. Furthermore, by placing the polarized crystal in a neutron optics focus and de-focus scheme, the actual sample cross-section could be increased by a factor 35 corresponding to an effective spin filter cross-section of 18×18mm2.
Apart from their very classical use to build polarized targets for particle physics, the methods of dynamic nuclear polarization (DNP) have more recently found application for sensitivity enhancement ...in high-resolution NMR, both in the solid and in the liquid state. It is often thought that the possible signal enhancement in such applications deteriorates when the DNP is performed at higher fields. We show that for a dissolution-DNP method that uses conventional (2,2,6,6-tetramethylpiperidine 1-oxyl) radicals as the paramagnetic agent, this is not the case for fields up to 5 T.
A novel neutron spin filter whose principle is based on the strong spin dependence of the neutron scattering on protons has been developed. The dimensions of this filter are small, and it works very ...efficiently and is stable even in inhomogeneous fields. The protons in the naphthalene spin filter crystal are polarized by a recently developed method of dynamic nuclear polarization using photoexcited triplet states. This technique allows the design of a very compact apparatus that can be placed at a close distance to the sample under investigation. The application of this filter as a polarization analyzer is demonstrated in a magnetic small‐angle neutron scattering experiment with the measurement of the spin‐dependent scattering signals of a CuFeNi alloy. This sample has a pronounced textured structure factor of ferromagnetic precipitates in a paramagnetic matrix. The performance of the spin filter as an analyzer is illustrated by the excellent agreement of the experimental data with simulations based on a model of homogeneously magnetized spherical particles which are ordered in a simple cubic paracrystalline lattice.
Display omitted
•Apparatus for pulse X-band ESR, pulse DNP and NMR with optically excited triplet states.•Helium flow cryostat with fiber coupling stage to high power laser system.•Large diameter ...dielectric ring ESR probe with integrated NMR coil.•High proton spin polarization above 50% in bulk samples at 0.3T and 100K.
In standard Dynamic Nuclear Polarization (DNP) electron spins are polarized at low temperatures in a strong magnetic field and this polarization is transferred to the nuclear spins by means of a microwave field. To obtain high nuclear polarizations cryogenic equipment reaching temperatures of 1K or below and superconducting magnets delivering several Tesla are required. This equipment strongly limits applications in nuclear and particle physics where beams of particles interact with the polarized nuclei, as well as in neutron scattering science. The problem can be solved using short-lived optically excited triplet states delivering the electron spin. The spin is polarized in the optical excitation process and both the cryogenic equipment and magnet can be simplified significantly. A versatile apparatus is described that allows to perform pulsed dynamic nuclear polarization experiments at X-band using short-lived optically excited triplet sates. The efficient 4He flow cryostat that cools the sample to temperatures between 4K and 300K has an optical access with a coupling stage for a fiber transporting the light from a dedicated laser system. It is further designed to be operated on a neutron beam. A combined pulse ESR/DNP spectrometer has been developed to observe and characterize the triplet states and to perform pulse DNP experiments. The ESR probe is based on a dielectric ring resonator of 7mm inner diameter that can accommodate cubic samples of 5mm length needed for neutron experiments. NMR measurements can be performed during DNP with a coil integrated in the cavity. With the presented apparatus a proton polarization of 0.5 has been achieved at 0.3T.
In dynamic nuclear polarisation (DNP), also called hyperpolarisation, a small amount of unpaired electron spins is added to the sample containing the nuclear spins, and the polarisation of these ...unpaired electron spins is transferred to the nuclear spins by means of a microwave field. Traditional DNP polarises the electron spin of stable paramagnetic centres by cooling down to low temperature and applying a strong magnetic field. Then weak continuous wave microwave fields are used to induce the polarisation transfer. Complicated cryogenic equipment and strong magnets can be avoided using short-lived photo-excited triplet states that are strongly aligned in the optical excitation process. However, a much faster transfer of the electron spin polarisation is needed and pulsed DNP methods like nuclear orientation via electron spin locking (NOVEL) and the integrated solid effect (ISE) are used.
To describe the polarisation transfer with the strong microwave fields in NOVEL and ISE, the usual perturbation methods cannot be used anymore. In the previous paper, we presented a theoretical approach to calculate the polarisation transfer in ISE. In the present paper, the theory is applied to the system naphthalene-h
8
doped with pentacene-d
14
yielding the photo-excited triplet states and compared with experimental results.
We report experiments on the production of ultracold neutrons (UCN) in a converter of superfluid helium coated with fluorinated grease (fomblin). We employed our special technique of window-free ...extraction of accumulated UCN from the superfluid helium, in which they were produced by downscattering neutrons of a cold beam from the Munich research reactor. The fomblin-coating reduced the time constant for UCN passage through the extraction hole by a factor three compared to our previous experiment employing an uncoated stainless steel vessel. A time-of-flight measurement of the cold neutron spectrum incident on the converter, combined with a gold foil activation, allowed us to determine both the single-phonon and multi-phonon contributions to the UCN production. The UCN production rate is in reasonable agreement with the theoretical expectation.
We report on a novel neutron radiography technique that uses the Ramsey principle, a method similar to neutron spin echo. For the first time quantitative imaging measurements of magnetic objects and ...fields could be performed. The strength of the spin-dependent magnetic interaction is detected by a change in the Larmor precession frequency of the neutron spins. Hence, one obtains in addition to the normal attenuation radiography image a so-called neutron spin phase image, which provides a two-dimensional projection of the magnetic field integrated over the neutron flight path.
PDF
