Biological Solids Research Articles

Slow global motions in biosolids studied by the deuteron stimulated echo NMR experiment.

Recent 15N R1ρ-relaxation studies have shown that proteins in the solid state undergo slow, low amplitude global motion in the sub-millisecond time range. This range is at the edge of the time window for R1ρ experiments and, therefore, the motional parameters obtained by this method are not precise or reliable. In this paper, we present a 2H stimulated echo study of this type of molecular dynamics. The 2H stimulated echo experiments on a static sample allow for direct measurement of the correlation function in the time range of 10-6-10-1s, making them well suited to study this type of molecular mobility. We have conducted a detailed analytical and numerical comparison of the correlation functions obtained from the relaxation and stimulated echo experiments, which are generally different. We have identified conditions and algorithms that enable a direct comparison of the relaxation and stimulated echo experimental results. Using the protein GB1 in the form of a lyophilized powder, we have demonstrated that 15N R1ρ-relaxation and 2H stimulated echo experiments yield essentially the same slow-motion correlation function. Surprisingly, this type of motion is observed not only in the protein sample but also in the tripeptide and single amino acid solid samples. The comparison of data measured in these three samples at different temperatures led us to conclude that this slow motion is, in fact, ultrasonic phonons, which seem to be inherent to all rigid biological solids.

Acclimatisation process of biogas production from tofu industrial wastewater using biofilter in anaerobic baffled reactor (ABR)

A biofilter is a simple technology used in an anaerobic baffled reactor (ABR) to keep biological solids (inoculum) from being easily carried by the inlet substrate and to shorten the hydraulic retention time (HRT). The principle of a biofilter is to form a biofilm on the packed bed of the biofilter in ABR so that it contains immobilised microorganisms. This study aims to know the performance of the biofilter reactor on biogas production during the acclimatisation process. The results show biofilters shorten the HRT and effectively remove pollutants, increasing biogas production and methane quality. The total solid content decreases by around 44 %, from 0.38 % to 0.17 %. The biogas production during acclimatisation was 1806.41 L and COD removal was 95 %. The biogas composition of CH4 was 58.05 %, CO2 38.23 %, and N2 3.2 %. This study provides preliminary findings for further studies on the use of tofu wastewater as a biogas feedstock with different concentrate substrates, which is very useful for sustainability activities and improving the industry to become green.

17O Solid-State NMR Spectroscopy of Lipid Membranes.

Despite the limitations posed by poor sensitivity, studies have reported the unique advantages of 17O based NMR spectroscopy to study systems existing in liquid, solid, or semisolid states. 17O NMR studies have exploited the remarkable sensitivity of quadrupole coupling and chemical shift anisotropy tensors to the local environment in the characterization of a variety of intra- and intermolecular interactions and motion. Recent studies have considerably expanded the use of 17O NMR to study dynamic intermolecular interactions associated with some of the challenging biological systems under magic angle spinning (MAS) and aligned conditions. The very fast relaxing nature of 17O has been well utilized in cellular and in vivo MRS (magnetic resonance spectroscopy) and MRI (magnetic resonance imaging) applications. The main focus of this Review is to highlight the new developments in the biological solids with a detailed discussion for a few selected examples including membrane proteins and nanodiscs. In addition to the unique benefits and limitations, the remaining challenges to overcome, and the impacts of higher magnetic fields and sensitivity enhancement techniques are discussed.

A primer to polarizing agent design: Quantum mechanical understanding of cross effect magic-angle spinning Dynamic Nuclear Polarization

Dynamic Nuclear Polarization (DNP) is transforming the landscape of solid-state characterization for both biological solids and functional materials. By transferring electron spin polarization to coupled nuclear spins under microwave irradiation, DNP increases NMR sensitivity by several orders of magnitude. However, the mechanism of DNP transfer and its efficiency under magic-angle spinning (MAS) significantly differs from that under static conditions. This primer article provides a comprehensive and pedagogical explanation of the theoretical aspects of MAS-DNP, with a specific focus on the cross-effect mechanism. A clear understanding of the nuances of MAS-DNP is crucial for improving its efficiency and extending its application to high magnetic fields and fast MAS conditions. To this end, the article proposes a guideline for synthetic chemists to develop DNP polarizing agents under these experimental conditions.

Fast magic angle spinning for the characterization of milligram quantities of organic and biological solids at natural isotopic abundance by 13C–13C correlation DNP-enhanced NMR

We show that multidimensional solid-state NMR 13C–13C correlation spectra of biomolecular assemblies and microcrystalline organic molecules can be acquired at natural isotopic abundance with only milligram quantities of sample. These experiments combine fast Magic Angle Spinning of the sample, low-power dipolar recoupling, and dynamic nuclear polarization performed with AsymPol biradicals, a recently introduced family of polarizing agents. Such experiments are essential for structural characterization as they provide short- and long-range distance information. This approach is demonstrated on diverse sample types, including polyglutamine fibrils implicated in Huntington's disease and microcrystalline ampicillin, a small antibiotic molecule.

Editorial: theme issue on complex rheology in biological systems

Editorial: theme issue on complex rheology in biological systems

Solid-State NMR: Methods for Biological Solids.

In the last two decades, solid-state nuclear magnetic resonance (ssNMR) spectroscopy has transformed from a spectroscopic technique investigating small molecules and industrial polymers to a potent tool decrypting structure and underlying dynamics of complex biological systems, such as membrane proteins, fibrils, and assemblies, in near-physiological environments and temperatures. This transformation can be ascribed to improvements in hardware design, sample preparation, pulsed methods, isotope labeling strategies, resolution, and sensitivity. The fundamental engagement between nuclear spins and radio-frequency pulses in the presence of a strong static magnetic field is identical between solution and ssNMR, but the experimental procedures vastly differ because of the absence of molecular tumbling in solids. This review discusses routinely employed state-of-the-art static and MAS pulsed NMR methods relevant for biological samples with rotational correlation times exceeding 100's of nanoseconds. Recent developments in signal filtering approaches, proton methodologies, and multiple acquisition techniques to boost sensitivity and speed up data acquisition at fast MAS are also discussed. Several examples of protein structures (globular, membrane, fibrils, and assemblies) solved with ssNMR spectroscopy have been considered. We also discuss integrated approaches to structurally characterize challenging biological systems and some newly emanating subdisciplines in ssNMR spectroscopy.

Proton-detected 15N-1H dipolar coupling/1H chemical shift correlation experiment for the measurement of NH distances in biological solids under fast MAS solid-state NMR

Measurement of distances from dipolar couplings is essential for structural characterization, refinement and validation using the solid-state nuclear magnetic resonance (ssNMR) spectroscopy. Particularly, knowledge about NH dipolar interactions in biological solids is important for understanding the hydrogen (H)-bonding interactions, molecular geometry and spin dynamics. In this regard, we have proposed a proton-detected two-dimensional (2D) 15N-1H dipolar coupling/1H chemical shift correlation experiment using the C-symmetry based windowless recoupling of chemical shift anisotropy (ROCSA) in combination with the DIPSHIFT pulse-based method for the measurement of short NH distances in the isotopically labeled and naturally abundant biological solids at fast magic angle spinning (MAS) rates (40–70 kHz). Our proposed method results in undistorted recoupled 15N-1H dipolar coupling powder lineshapes that are free from the recoupled 1H CSA contributions under the 15N evolution, a feature that is essential for the measurement of NH distances with improved accuracy (± 500 Hz in terms of the NH dipolar couplings). The pulse sequence developed in the present study is also insensitive to the 1H–1H homonuclear dipolar interactions, relaxation effects owing to its constant-time implementation, and t1-noise from the fluctuations in the MAS.

Evaluation of freshwater prawn, Macrobrachium rosenbergii, for biological solids control in raft aquaponic systems and the protective effectiveness of root guards

AbstractThe accumulation of excess solids within the hydroponic troughs of raft aquaponic systems is considered one of the most common problems in commercial production. These solids can negatively affect water quality during decomposition. They can also impact plant health by physically coating the plant roots, reducing nutrient uptake and inducing plant diseases such as Pythium. Manual removal is labor intensive and costly. Based on their detritivorous feeding behavior, the freshwater prawn, Macrobrachium rosenbergii, may have potential as a biological control of solids. However, in a preliminary trial it was found that prawn will damage unprotected plant roots. To address this, two trials were conducted to evaluate the impact of prawn on solids accumulation within the plant beds and the effectiveness of different mechanical approaches to protect the plant roots. Both trials used six replicate recirculating raft aquaponic systems. Three systems had no prawn and three had prawn (~8 g) stocked at five prawn per trough. The plant grown was Buttercrunch Bibb lettuce (Lactuca sativa) at a density of 24 plants/m2. Plant growth in Trial 1 was statistically analyzed using a 2 × 3 factorial with the main effects of presence of prawn (none or 5/trough) and root protection device (none, plastic screen, or cloth netting). Water quality and solids data were compared in systems with prawn and without prawn using a two sample t test. Trial 1 lasted 32 days. Trial 2 used a plastic mesh “false bottom” to exclude prawn from access to the plants over the entire beds. Trial 2 used six systems all with the false bottoms in place. The comparison of three systems with prawn and three systems without prawn and was conducted over two plant production cycles of 4 weeks each. All data was analyzed using a two sample t test. In Trial 1, the presence of prawn produced a highly significant (p ≤ 0.01) reduction of settled solid wastes within the troughs, but plant growth was significantly reduced (p ≤ 0.01) in beds stocked with prawn regardless of root protection device. In Trial 2, the presence of prawn again produced a highly significant (p ≤ 0.01) reduction of settled solid waste within the troughs. There was no significant difference (p > 0.05) in average plant weight with this root protection device in systems with or without prawn. This research demonstrates that freshwater prawn can reduce solid waste buildup in aquaponic plant beds, but that effective root protection is essential to maintain efficient plant production.

Estimation of anisotropic parameters in liquid crystals using a constant time NMR method

A method for measurement of anisotropic parameters like 1H chemical shifts and the estimation of 1H chemical shift anisotropy (CSA) in liquid crystals is presented. The estimation of 1H CSA is essential in order to understand the structural information of several systems like membrane proteins, organic and biological solids. After obtaining the information of chemical shifts in the liquid crystalline phase, alignment-induced shifts (AIS) are calculated. The dependence of AIS values and the local order parameters and CSA can be utilized to calculate the 1H CSA liquid crystals. The advantage with proton chemical shifts is that, it is sensitive not only to local structural details but also to the inter-molecular interactions. This could be an advantage in the study of systems like solids and liquid crystals.

Improved NMR transfer of magnetization from protons to half-integer spin quadrupolar nuclei at moderate and high magic-angle spinning frequencies.

Half-integer spin quadrupolar nuclei are the only magnetic isotopes for the majority of the chemical elements. Therefore, the transfer of polarization from protons to these isotopes under magic-angle spinning (MAS) can provide precious insights into the interatomic proximities in hydrogen-containing solids, including organic, hybrid, nanostructured and biological solids. This transfer has recently been combined with dynamic nuclear polarization (DNP) in order to enhance the NMR signal of half-integer quadrupolar isotopes. However, the cross-polarization transfer lacks robustness in the case of quadrupolar nuclei, and we have recently introduced as an alternative technique a -RINEPT (through-space refocused insensitive nuclei enhancement by polarization transfer) scheme combining a heteronuclear dipolar recoupling built from adiabatic pulses and a continuous-wave decoupling. This technique has been demonstrated at 9.4 T with moderate MAS frequencies, -15 kHz, in order to transfer the DNP-enhanced H polarization to quadrupolar nuclei. Nevertheless, polarization transfers from protons to quadrupolar nuclei are also required at higher MAS frequencies in order to improve the H resolution. We investigate here how this transfer can be achieved at and 60 kHz. We demonstrate that the -RINEPT sequence using adiabatic pulses still produces efficient and robust transfers but requires large radio-frequency (rf) fields, which may not be compatible with the specifications of most MAS probes. As an alternative, we introduce robust and efficient variants of the -RINEPT and PRESTO (phase-shifted recoupling effects a smooth transfer of order) sequences using symmetry-based recoupling schemes built from single and composite pulses. Their performances are compared using the average Hamiltonian theory and experiments at T on -alumina and isopropylamine-templated microporous aluminophosphate (AlPO-14), featuring low and significant H-H dipolar interactions, respectively. These experiments demonstrate that the H magnetization can be efficiently transferred to Al nuclei using -RINEPT with (27090) recoupling and using PRESTO with (180) or (27090) schemes at or 62.5 kHz, respectively. The -RINEPT and PRESTO recoupling schemes complement each other since the latter is affected by dipolar truncation, whereas the former is not. We also analyze the losses during these recoupling schemes, and we show how these magnetization transfers can be used at kHz to acquire in 72 min 2D HETCOR (heteronuclear correlation) spectra between H and quadrupolar nuclei, with a non-uniform sampling (NUS).

Efficient symmetry-based γ-encoded DQ recoupling sequences for suppression of t1-noise in solid-state NMR spectroscopy at fast MAS

Solid-state NMR spectroscopy has played a significant role in elucidating the structure and dynamics of materials and biological solids at a molecular level for decades. In particular, the 1H double-quantum/single-quantum (DQ/SQ) chemical shift correlation experiment is widely used for probing the proximity of protons, rendering it a powerful tool for elucidating the hydrogen-bonding interactions and molecular packing of various complex molecular systems. Two factors, namely, the DQ filtering efficiency and t1-noise, dictate the quality of the 2D 1H DQ/SQ spectra. Experimentally different recoupling sequences show varied DQ filtering efficiencies and t1-noise. Herein, after a systematic search of symmetry-based DQ recoupling sequences, we report that the symmetry-based γ-encoded RNnν sequences show superior performance to other DQ recoupling sequences, which not only have a higher DQ recoupling efficiency but can also significantly reduce t1-noise. The origin of t1-noise is further discussed in detail via extensive numerical simulations. We envisage that such γ-encoded RNnν sequences are superior candidates for DQ recoupling in proton-based solid-state NMR spectroscopy due to its capability of efficiently exciting DQ coherences and suppressing t1-noise.

Broadband adiabatic inversion cross-polarization to integer-spin nuclei with application to deuterium NMR.

Solid-state NMR (SSNMR) spectroscopy of integer-spin quadrupolar nuclei is important for the molecular-level characterization of a variety of materials and biological solids; of the integer spins, 2 H (S = 1) is by far the most widely studied, due to its usefulness in probing dynamical motions. SSNMR spectra of integer-spin nuclei often feature very broad powder patterns that arise largely from the effects of the first-order quadrupolar interaction; as such, the acquisition of high-quality spectra continues to remain a challenge. The broadband adiabatic inversion cross-polarization (BRAIN-CP) pulse sequence, which is capable of cross-polarization (CP) enhancement over large bandwidths, has found success for the acquisition of SSNMR spectra of integer-spin nuclei, including 14 N (S = 1), especially when coupled with Carr-Purcell/Meiboom-Gill pulse sequences featuring frequency-swept WURST pulses (WURST-CPMG) for T2 -based signal enhancement. However, to date, there has not been a systematic investigation of the spin dynamics underlying BRAIN-CP, nor any concrete theoretical models to aid in its parameterization for applications to integer-spin nuclei. In addition, the BRAIN-CP/WURST-CPMG scheme has not been demonstrated for generalized application to wideline or ultra-wideline (UW) 2 H SSNMR. Herein, we provide a theoretical description of the BRAIN-CP pulse sequence for spin-1/2 → spin-1 CP under static conditions, featuring a set of analytical equations describing Hartmann-Hahn matching conditions and numerical simulations that elucidate a CP mechanism involving polarization transfer, coherence exchange, and adiabatic inversion. Several experimental examples are presented for comparison with theoretical models and previously developed integer-spin CP methods, demonstrating rapid acquisition of 2 H NMR spectra from efficient broadband CP.

Novel formulations and drug delivery systems to administer biological solids.

Recent advances in formulation sciences have expanded the previously limited design space for biological modalities, including peptide, protein, and vaccine products. At the same time, the discovery and application of new modalities, such as cellular therapies and gene therapies, have presented formidable challenges to formulation scientists. We explore these challenges and highlight the opportunities to overcome them through the development of novel formulations and drug delivery systems as biological solids. We review the current progress in both industry and academic laboratories, and we provide expert perspectives in those settings. Formulation scientists have made a tremendous effort to accommodate the needs of these novel delivery routes. These include stability-preserving formulations and dehydration processes as well as dosing regimes and dosage forms that improve patient compliance.

Pharmaceutical protein solids: Drying technology, solid-state characterization and stability.

Despite the boom in biologics over the past decade, the intrinsic instability of these large molecules poses significant challenges to formulation development. Almost half of all pharmaceutical protein products are formulated in the solid form to preserve protein native structure and extend product shelf-life. In this review, both traditional and emerging drying techniques for producing protein solids will be discussed. During the drying process, various stresses can impact the stability of protein solids. However, understanding the impact of stress on protein product quality can be challenging due to the lack of reliable characterization techniques for biological solids. Both conventional and advanced characterization techniques are discussed including differential scanning calorimetry (DSC), solid-state Fourier transform infrared spectrometry (ssFTIR), solid-state fluorescence spectrometry, solid-state hydrogen deuterium exchange (ssHDX), solid-state nuclear magnetic resonance (ssNMR) and solid-state photolytic labeling (ssPL). Advanced characterization tools may offer mechanistic investigations into local structural changes and interactions at higher resolutions. The continuous exploration of new drying techniques, as well as a better understanding of the effects caused by different drying techniques in solid state, would advance the formulation development of biological products with superior quality.

Expanding the NMR toolkit for biological solids: oxygen-17 enriched Fmoc-amino acids

We report the solid-state 17O NMR parameters for five previously uncharacterized N-α-fluoren-9-yl-methoxycarbonyl-O-t-butyl (Fmoc) protected amino acids.

A mathematical framework for optimum design and operation of SBR processes

The operational flexibility of Sequencing Batch Reactors (SBR) makes this technology especially resilient to changes in functional requirements. A main feature of SBRs is that plant upgrading from nitrogen to phosphorus removal specifications becomes straightforward by simple adaptation of the manipulated variables. In contrast, the optimum design and operation of these processes is an intricate problem whose formulation involves multiple considerations such as mathematical descriptions for both biological transformations and solids separation phenomena, design procedures for air diffusers, etc. Moreover, the dynamic nature of SBRs adds extra complexity when it comes to solving optimization problems for this technology. In this paper, a model-based optimization framework is proposed to solve the optimum design and operation of SBR systems. This framework splits the whole problem into two single objective optimization sub-problems. The dimensions of the SBR are first calculated; Then, the operational costs in the long-term are optimized. A case-study for a population of 100000 inhabitant-equivalent demonstrates the potential of the framework.

Impact of Magnetic Field Strength on Resolution and Sensitivity of Proton Resonances in Biological Solids

Sensitivity and resolution together determine the quality of NMR spectra in biological solids. Higher magic angle spinning frequencies yield a more efficient suppression of the coupling network and...

Multivariate analysis of activated sludge community in full-scale wastewater treatment plants

We investigated changes in protozoa and metazoa community in relation to process parameters in activated sludge from four wastewater treatment plants (WWTPs) throughout the period of 1 year. Principal component analysis (PCA) showed that activated sludge from investigated treatment plants had different dominating species representatives and community composition mainly depends on individual features of the treatment plants. Redundancy analysis (RDA) showed that the temperature in bioreactors was the most relevant factor explaining changes in the microorganism community, whereas reduction rate of chemical oxygen demand (COD), biological oxygen demand (BOD5), suspended solids (SS), and total nitrogen (TN) did not sufficiently explain the variation in protozoa and metazoan community composition. The results indicate that in stable working WWTP it is difficult to find a pronounced link between activated sludge species composition, process parameters, and plant configuration. Applied multivariate analysis can be a valuable tool for the exploration of the relations between community composition and WWTP process parameters.

Sustainable management of biological solids in small treatment plants: overview of strategies and reuse options for a solar drying facility in Poland

The issue of sustainable management of biosolids (excess sludge) from wastewater treatment is an important issue in the entire developed world. Residual sludge disposal costs and environmental impact may be significant, and reducing such costs, as well as the energy consumption for dewatering and drying, is a key issue for safe and sustainable sludge disposal, considering the recent ban of some disposal options, such as landfilling, in many European countries. An alternative to thermal technologies is solar drying (not to be confused with bio-drying, very close to the concept of composting). Solar greenhouse drying technology is characterized by reduced land requirements compared with traditional outdoor drying beds, as well as by low-energy requirements compared with other thermal drying methods. Process operation is cost-efficient, with close to no maintenance, and observed specific evaporation rates up to threefold higher than conventional drying beds. Many applications of this technology exist in Poland, Germany and Austria: more than 10,000 t of wet sludge per year is treated in this way in Germany alone and almost as many (9000 t/year) in Poland. This paper examines current biosolids treatment technologies applicable to small wastewater treatment plants (2000–9999 population equivalents served) and opportunities for possible solids reuse in Poland in view of sustainable circular economy schemes. In particular, a purely solar-driven greenhouse facility for sewage sludge drying was investigated under different conditions (season, temperature, environmental humidity) and possible improvements for its efficiency evaluated. Sludge processed by solar drying could have different final disposal pathways, according to season, in accordance with the prescriptions of the new National Waste Management Plan of Poland.