Optimising sample preparation for FTIR-based microplastic analysis in wastewater and sludge samples: multiple digestions

The lack of standardised methodologies in microplastic research has been addressed in recent years as it hampers the comparison of results across studies. The quantification of microplastics in the environment is key to the assessment of the potential eco-toxicological impacts that this new category of emerging pollutants could have on terrestrial and aquatic species. Therefore, the need for protocols that are robust, simple and reliable together with their standardisation are of crucial importance. This study has focused on removal of organic matter with Fenton reagent from wastewater and sludge samples. This step of analysis was optimised by implementing a multi-digestion treatment on these samples that have high concentration of complex mixtures of organic matter, which interfere with microplastic enumeration. Moreover, this study targeted the detection of microplastics in the sub-hundred-micron size range due to the potential higher risks associated with smaller-sized particles and the limited data available from previous wastewater research. To show the validity of the method, triplicate samples of raw sewage, final effluent and sludge were independently spiked with two different sizes and types of microplastic polymers. Due to the various analytical stages required for the isolation of microplastics, time is a limiting factor in sample processing. The sequential digestion with Fenton reagent represents an inexpensive and time-efficient procedure for wastewater research providing effective degradation of organic material. These advantages over other currently available methods mean the method is suitable for analysis of large numbers of samples allowing robust monitoring data sets to be generated.

Primary culture of chondrocytes after collagenase IA or II treatment of articular cartilage from elderly patients undergoing arthroplasty

Background Joint replacement surgery provides articular cartilage samples for chondrocyte isolation. To our knowledge, the effect of the collagenase type on releasing of chondrocytes from the extracellular matrix of cartilage is not reported. Objectives To determine whether cartilage digested with collagenase IA yielded more chondrocytes than that digested with collagenase II and determine whether chondrocytes isolated with collagenase IA could be cultured in vitro. Methods Cartilage slices collected from 18 elderly patients who received joint replacement surgery (16 hips, 2 knees) were digested sequentially with 0.4% pronase E and 0.02% collagenase IA, or with 0.15% collagenase II alone, or sequentially with 0.4% pronase E and 0.02% collagenase II. We compared cell yield from each method. Cell viability by the most effective method was calculated and plotted. The morphology of cultured monolayer chondrocytes was recorded with a light microscope. Results Sequential digestion with pronase E and collagenase IA yielded 2566 ± 873 chondrocytes per mg wet cartilage, which was more effective than the other isolation methods (P = 0.018). The average chondrocyte viability could reach 84% ± 8% (n = 11). Light microscopic images showed typical chondrocyte morphology in monolayer cultures. Conclusion Sequential digestion of human articular cartilage with pronase E and collagenase IA was more effective than collagenase II alone or collagenase II combined with pronase E for releasing chondrocytes from extracellular matrix of cartilage. Chondrocytes isolated with this method could be maintained in monolayer cultures for at least 2 passages with unaltered morphology.

Discovery of exolytic heparinases and their catalytic mechanism and potential application

Heparinases (Hepases) are critical tools for the studies of highly heterogeneous heparin (HP)/heparan sulfate (HS). However, exolytic heparinases urgently needed for the sequencing of HP/HS chains remain undiscovered. Herein, a type of exolytic heparinases (exoHepases) is identified from the genomes of different bacteria. These exoHepases share almost no homology with known Hepases and prefer to digest HP rather than HS chains by sequentially releasing unsaturated disaccharides from their reducing ends. The structural study of an exoHepase (BIexoHep) shows that an N-terminal conserved DUF4962 superfamily domain is essential to the enzyme activities of these exoHepases, which is involved in the formation of a unique L-shaped catalytic cavity controlling the sequential digestion of substrates through electrostatic interactions. Further, several HP octasaccharides have been preliminarily sequenced by using BIexoHep. Overall, this study fills the research gap of exoHepases and provides urgently needed tools for the structural and functional studies of HP/HS chains.

Discovery of exolytic heparinases and their catalytic mechanism and potential application

Heparinases are critical tools for the studies of highly heterogeneous heparin (HP)/heparan sulfate (HS). However, the exolytic heparinases urgently needed for the sequencing of HP/HS chains are seemingly inaccessible. Herein, a type of exolytic heparinases (exoHepases) was identified from the genomes of different bacteria. These exoHepases share almost no homology with known Hepases and prefer to digest HP rather than HS chains by sequentially releasing unsaturated disaccharides from their reducing ends. The structural study of an exoHepase (exoHep) shows that an N-terminal conserved DUF4962 superfamily domain is essential to the exolytic activity of these exoHepases, which is involved in the formation of a unique L-shaped catalytic cavity controlling the sequential digestion of substrates through electrostatic interactions. Further, several HP octasaccharides were preliminarily sequenced by using exoHep. Overall, this study fills the research gap of exoHepases and provides urgently needed tools for the structural and functional studies of HP/HS chains.

Co-Digestion of Salix and Manure for Biogas: Importance of Clone Choice, Coppicing Frequency and Reactor Setup

Animal manure represents a major source of renewable energy that can be converted into biogas using anaerobic digestion. In order to most efficiently utilize this resource, it can be co-digested with energy dense, high biomethanation potential feedstocks such as energy crops. However, such feedstocks typically require pretreatments which are not feasible for small-scale facilities. We investigated the use of single-stage and the sequential co-digestion of comminuted but otherwise non-pretreated Salix with animal manure, and further investigated the effects of coppicing frequency and clone choice on biomethanation potential and the area requirements for a typical Swedish farm-scale anaerobic digester using Salix and manure as feedstock. In comparison with conventional single-stage digestion, sequential digestion increased the volumetric and specific methane production by 57% to 577 NmL L−1 d−1 and 192 NmL (g volatile solids (VS))−1, respectively. Biomethanation potential was the highest for the two-year-old shoots, although gains in biomass productivity suggest that every-third-year coppicing may be a better strategy for supplying Salix feedstock for anaerobic digestion. The biomethane production performance of the sequential digestion of minimally pretreated Salix mirrors that of hydrothermally pretreated hardwoods and may provide an option where such pretreatments are not feasible.

Leaching of Rare Earth Elements from Central Appalachian Coal Seam Underclays

Rare earth elements (REE) are necessary for advanced technological and energy applications. To support the emerging need, it is necessary to identify new domestic sources of REE and technologies to separate and recover saleable REE product in a safe and economical manner. Underclay rock associated with Central Appalachian coal seams and prevalent in coal utilization waste products is an alternative source of REE to hard rock ores that are mainly composed of highly refractory REE-bearing minerals. This study utilizes a suite of analytical techniques and benchtop leaching tests to characterize the properties and leachability of the coal seam underclays sampled. Laboratory bench-top and flow-through reactor leaching experiments were conducted on underclay rock powders to produce a pregnant leach solution (PLS) that has relatively low concentrations of gangue elements Al, Si, Fe, and Th and is amenable to further processing steps to recover and produce purified REE product. The leaching method described here uses a chelating agent, the citrate anion, to solubilize elements that are adsorbed, or weakly bonded to the surface of clay minerals or other mineral solid phases in the rock. The citrate PLS produced from leaching specific underclay powders contains relatively higher concentrations of REE and lower concentrations of gangue elements compared to PLS produced from sequential digestion using ammonium sulfate and mineral acids. Citrate solution leaching of underclay produces a PLS with lower concentrations of gangue elements and higher concentrations of REE than achieved with hydrochloric acid or sulfuric acid. The results provide a preliminary assessment of the types of REE-bearing minerals and potential leachability of coal seam underclays from the Central Appalachian basin.

Microencapsulation of Bifidobacterium animalis subsp. lactis BB-12 with mannitol

Bifidobacterium animalis subsp. lactis BB-12 (BB-12) was microencapsulated using co-extrusion technology with chitosan coating and the incorporation of mannitol as prebiotic. Optimization of coating material chitosan concentration (0–0.5% w/v) and mannitol concentration (0–5% w/v) as prebiotic were performed to determine the formulation that produces beads with desired properties. The microencapsulation efficiency (MEE) of free and microencapsulated BB-12 (with and without mannitol) were determined. All forms of BB-12 further subjected to sequential digestion in simulated gastric juice (SGJ, pH 2.0) for 2 hours and simulated intestinal juice (SIJ, pH 7.5) for 3 hours. The results indicated that 0.4% (w/v) of chitosan coating and 3% (w/v) of mannitol were the optimum concentrations to produce microencapsulated BB-12 with the highest MEE of 89.15% and the average bead size of 805 µm. The BB-12 beads produced through co-extrusion were spherical with a smooth surface. Throughout the five hours sequential gastrointestinal digestion, both microencapsulated BB-12 with and without mannitol were able to maintain their viable cell count at least 106 CFU/g at the end of the incubation. The presence of prebiotic mannitol showed a significant protective effect on the microencapsulated BB-12 during gastrointestinal transit.

Contributions of Chondroitin Sulfate, Keratan Sulfate and N-linked Oligosaccharides to Inhibition of Neurite Outgrowth by Aggrecan

The role of proteoglycans in the central nervous system (CNS) is a rapidly evolving field and has major implications in the field of CNS injury. Chondroitin sulfate proteoglycans (CSPGs) increase in abundance following damage to the spinal cord and inhibit neurite outgrowth. Major advances in understanding the interaction between outgrowing neurites and CSPGs has created a need for more robust and quantitative analyses to further our understanding of this interaction. We report the use of a high-throughput assay to determine the effect of various post-translational modifications of aggrecan upon neurite outgrowth from NS-1 cells (a PC12 cell line derivative). Aggrecan contains chondroitin sulfate, keratan sulfate, and N-linked oligosaccharides (N-glycans), each susceptible to removal through different enzymatic digestions. Using a sequential digestion approach, we found that chondroitin sulfate and N-glycans, but not keratan sulfate, contribute to inhibition of neurite outgrowth by substrate-bound aggrecan. For the first time, we have shown that N-linked oligosaccharides on aggrecan contribute to its inhibition of neuritogenesis.