Influence of the intensive mariculture on coastal sedimentary organic matter: Insight from size-fractionated particles.

A portion of carbon produced from shellfish and kelp cultivation is buried in sedimentary environment, and mariculture carbon sequestration potential is an important part of marine carbon sink and has attracted worldwide attention. Total organic carbon (TOC), total nitrogen (TN) and their stable isotopes (δ13C and δ15N), as well as the mass distribution of these size-fractionated particles were determined in order to study the distribution and sources of TOC in Sanggou Bay. Results showed that sediment organic matter has complex sources from kelp (30.4 %), marine phytoplankton (25.6 %), shellfish (23.7 %), terrestrial input (20.3 %), and mariculture activities of shellfish and kelp was the major component in surface sediment. Approximately 44-69 % of TOC was associated with the 16-32 µm fraction. Low δ13C (-22.1 to -15.1‰) and high δ15N (5.0-5.7‰) were observed in fine particles (<16 µm), indicating relatively high contribution of marine phytoplankton and mariculture derived organic carbon. On the contrary, relatively higher δ13C (-20.2 to -9.2‰) and lower values δ15N (-4.7 to 5.2‰) in coarse particles (>32 µm) suggested that sedimentary organic carbon might be influenced by some additional sources from terrestrial input or seaweed. The mass distribution, δ13C and δ15N of size-fractionated particles in sediments indicated that sediment was obviously redistributed under the condition of mariculture, and further suggested that mariculture derived organic matter have modified the distribution and sources of sedimentary organic matter. This study provided great insight into distribution and source of sedimentary organic carbon from the perspective of size-fractionated particles in mariculture area.

An integrated approach to vivianite recovery from waste activated sludge.

The waste activated sludge (WAS) of wastewater treatment system is often rich in phosphorus (P), which is a basic element of human life and could use up in the near future. This study proposed an integrated approach to efficiently recover P as vivianite from WAS and simultaneously enhance the sludge dewaterability. The raw WAS was first acidified using FeCl3, which was then fed to anaerobic fermenter for Fe3+ reduction. After fermentation, a technology named acid-elutriation was introduced to convert Fe and P from solid phase to liquid phase and concomitantly enhance the liquor-solid separation. Finally, vivianite was obtained via sludge eluate neutralization. The enhanced sludge dewaterability not only increases the recovery efficiency of Fe and P but also decreases the cost of sludge disposal.

Synchronization of Trypanosoma brucei by Counter-Flow Centrifugal Elutriation.

Centrifugal counter-flow elutriation is a non-invasive technique that separates cells based on their hydrodynamic volume in a specialized centrifugation chamber that allows the application of a counter-flow of buffer to oppose sedimentation. Here, we report a centrifugal counter-flow elutriation protocol for Trypanosoma brucei cells that is able to rapidly isolate highly enriched G1 subpopulations (>95%) of synchronized cells. The cells obtained are viable and proliferate without lag, allowing subsequent cell cycle phases to be obtained by continued culture. The synchronized cell cultures obtained by this process have uniform DNA content, a narrow size distribution, undergo synchronous division, and maintain synchrony into subsequent cell cycles.

Synchronization of Saccharomyces cerevisiae Cells for Analysis of Progression Through the Cell Cycle.

The cell division cycle is a fundamental process required for proliferation of all living organisms. The eukaryotic cell cycle follows a basic template with an ordered series of events beginning with G1 (Gap1) phase, followed successively by S (Synthesis) phase, G2 (Gap 2) phase, and M-phase (Mitosis). The process is tightly regulated in response to signals from both the internal and external milieu. The budding yeast S. cerevisiae is an outstanding model for the study of the cell cycle and its regulatory process. The basic events and regulatory processes of the S. cerevisiae cell cycle are highly conserved with other eukaryotes. The organism grows rapidly in simple medium, has a sequenced annotated genome, well-established genetics, and is amenable to analysis by proteomics and microscopy. Additionally, a range of tools and techniques are available to generate cultures of S. cerevisiae that are homogenously arrested or captured at specific phases of the cell cycle and upon release from that arrest these can be used to monitor cell cycle events as the cells synchronously proceed through a division cycle. In this chapter, we describe a series of commonly used techniques that are used to generate synchronized populations of S. cerevisiae and provide an overview of methods that can be used to monitor the progression of the cells through the cell division cycle.

An Optimized and Versatile Counter-Flow Centrifugal Elutriation Workflow to Obtain Synchronized Eukaryotic Cells.

Cell synchronization is a powerful tool to understand cell cycle events and its regulatory mechanisms. Counter-flow centrifugal elutriation (CCE) is a more generally desirable method to synchronize cells because it does not significantly alter cell behavior and/or cell cycle progression, however, adjusting specific parameters in a cell type/equipment-dependent manner can be challenging. In this paper, we used the unicellular eukaryotic model organism, Tetrahymena thermophila as a testing system for optimizing CCE workflow. Firstly, flow cytometry conditions were identified that reduced nuclei adhesion and improved the assessment of cell cycle stage. We then systematically examined how to achieve the optimal conditions for three critical factors affecting the outcome of CCE, including loading flow rate, collection flow rate and collection volume. Using our optimized workflow, we obtained a large population of highly synchronous G1-phase Tetrahymena as measured by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into nascent DNA strands, bulk DNA content changes by flow cytometry, and cell cycle progression by light microscopy. This detailed protocol can be easily adapted to synchronize other eukaryotic cells.

Purification of Basophils from Peripheral Human Blood.

The purification of basophils from peripheral blood has represented a formidable challenge for researchers since they were discovered by Paul Ehrlich in 1879. From the first published attempts in the late 1960s, it took half a century to develop robust protocols able to give sufficient numbers of pure, functionally unimpaired basophils. The existing protocols for basophil purification exploit those properties of basophils which distinguish them from other cell types such as their localization in blood, density, and the presence or absence of surface markers. Purification techniques have been used in various combinations and variations to achieve a common goal in mind: to obtain a pure population of human basophils in sufficient numbers for downstream studies. The arduous way leading up to the modern protocols is summarized in this historical retrospective. A fast protocol for purification of basophils to near homogeneity is also described.

Underpinning mechanistic understanding of the segregation phenomena of pharmaceutical blends using a near-infrared (NIR) spectrometer embedded segregation tester.

The segregation of an active pharmaceutical ingredient (API) within a powder blend is one of the major manufacturing obstacles in achieving content uniformity. Segregation can be due to differences in physicochemical properties of formulation components and/or perturbations experienced during secondary processing steps, such as granulation, fluidization, die-filling and compression. A near-infrared (NIR) spectrometer embedded segregation tester, which could mimic the external stimulations (vibration and fluidization) experienced by a blend in a manufacturing facility, was used to evaluate and predict blend segregation. Two different GlaxoSmithKline (GSK) product blends with variations in the API particle size and concentration were tested. Drug content was further measured at different locations along the powder bed by NIR to sketch the segregation profile and calculate the overall segregation intensity of each blend. The study indicated that the segregation potential was dependent on the particle sizes of API and excipients, as well as the type of stimulus applied (vibration vs fluidization). Drug concentration profiles obtained from this mode of analysis decoded the underlying segregation mechanisms (sieving, trajectory and air elutriation) easily. The employed NIR-based segregation tester proved to be a useful small-scale predictive tool to evaluate and rank the segregation risk of the studied pharmaceutical blends.

An integral approach to sludge handling in a WWTP operated for EBPR aiming phosphorus recovery: Simulation of alternatives, LCA and LCC analyses.

As phosphorus is a non-renewable resource mainly used to produce fertilizers and helps to provide food all over the world, the proper management of its reserves is a global concern since it is expected to become scarcer in the near future. In this work we assessed two different sludge line configurations aiming for P extraction and recovery before anaerobic digestion and compared them with the classical configuration. This study has been performed by simulation with the model BNRM2 integrated in the software package DESASS 7.1. Configuration 1 was based on the production of a PO4-enriched stream from sludge via elutriation in the primary thickeners, while Configuration 2 was based on the WASSTRIP® process and its PO4-enriched stream was mechanically obtained with dynamic thickeners. In both alternatives recovery was enhanced by promoting poly-phosphate (poly-P) extraction under anaerobic conditions, for which both configurations were fully evaluated in a full-scale WWTP. Both were also optimized to maximize phosphorus extraction. Their costs and life cycles were also analysed. The novelty of this research lies in the lack of literature about the integral evaluation of pre-anaerobic digestion P recovery from wastewaters. This study included a holistic approach and an optimization study of both alternatives plus their economic and environmental aspects. In Configuration 1, the PO4-P load in the recovery stream reached 43.1% of the total influent P load and reduced uncontrolled P-precipitation in the sludge line up to 52.9%. In Configuration 2, extraction was 48.2% of the influent P load and it reduced precipitation by up to 60.0%. Despite Configuration 1's lower phosphorus recovery efficiency, it had a 23.0% lower life cycle cost and a 14.2% lower global warming impact per hm3 of treated influent than Configuration 2. Configuration 1 also reduced the TAEC by 17.6% and global warming impact by 2.0% less than Configuration 0.

Hydrodynamic sorting controls the transport and hampers source identification of terrigenous organic matter: A case study in East China Sea inner shelf and its implication.

Transport of terrigenous organic matter (TerOM) in marginal seas plays an important role in marine carbon cycle which is closely related to global climate change. Suspended particulates in East China Sea (ECS) inner shelf are subject to strong influence of seasonally varied Zhe-Min Coastal Current (ZMCC) and Taiwan Warm Current (TWC). Transport of TerOM attached to the mineral particulates is therefore largely dependent on these hydrodynamic conditions. To address the transport pattern of TerOM in highly dynamic marginal seas and its implication, sediment samples were collected from ECS inner shelf and fractionated by water elutriation to simulate the hydrodynamic sorting process. Lignin phenols were determined for each fraction. The abundance of C phenols preferentially concentrated in the finer fractions, which is believed as a collective effect of hydrodynamic sorting, diagenetic reactivity and association preference of C phenols with finer sediments. In contrast to the expectation, sediments at the southernmost site did not have the highest mass percentage for the finest fraction, the lowest lignin abundance and the highest degradation status. Combining the results of lignin parameters with seawater temperature and salinity profiles, it is proposed that sediments are transported along the inner shelf southward in winter and northward in summer to some extent with the influence of seasonally varied ZMCC and TWC, induced by the seasonality of East Asia Monsoon (EAM). Finer grained sediments are more susceptible to this process. This transport pattern, which was only reported previously in ocean modeling, was firstly supported by the observation of lignin biomarkers. It is suggested that cross-shelf transport of sediments in ECS seems to be possible, but is complicated and is also affected by the seasonal variation of EAM. This study improved the understanding of hydrodynamic sorting on the transport of TerOM and carbon cycling in the marine system.

Effect of mechanical elutriation on carbon source recovery from primary sludge in a novel activated primary tank.

A novel activated primary tank (APT) with an elutriation unit was developed for recovering carbon by the fermentation and elutriation of primary sludge, and the mechanical elutriation mechanism was analysed by conducting a batch fermentation experiment to improve carbon source recovery. The results indicated that a high stirring velocity gradient could cause sludge disintegration, which could not only shorten the fermentation time, but also increase the production of soluble chemical oxygen demand (SCOD) and volatile fatty acids (VFAs) by 8.3% and 9.5%, respectively. Moreover, mechanical elutriation could also promote the release of SCOD from sludge to water, resulting in an increase in the yield of SCOD by 9.2%, it was observed that elutriation intensity plays a more important role than the elutriation time. The microbial community structure of the fermentation system was influenced by the stirring intensity. The relative abundance of fermentative bacteria in the reactor with a stirring intensity (G) of 160 s-1 was 13.8%, which was significantly higher than that in the reactor with G = 31 s-1 (8.037%), so the accumulation of VFAs and SCOD in the reactor with G = 160 s-1 was improved.

Characterizing the settleability of grit particles.

Grit chambers are installed at the headworks of a water resource recovery facility (WRRF) to reduce the impact of grit particles to the equipment and processes downstream. This settling process should thus be designed and operated in an efficient way. Despite the importance of knowing settling characteristics for design and operation of grit chambers, previous grit definitions have been based only on particle size characteristics, and not on settling velocities. Thus, this study presents an evaluation of the performance of two promising settling velocity characterization methods, ViCAs and elutriation, to characterize wastewater particles in view of the design and the optimization of the efficiency of the grit removal unit. PRACTITIONER POINTS: Settling characteristics are the governing parameter for grit chamber design. Since grit particles are vastly heterogeneous, it is preferred to measure these characteristics directly rather than to estimate them from particle size (and assumptions of density, form factor, …). More detailed settling information about grit particles can improve grit chamber design and estimation of removal performance. Adapted ViCAs and elutriation methods for faster settling particles allow studying the particle settling characteristics in a grit chamber. These methods are simple, fast, and cheap and only require small wastewater samples. A relationship was found between the influent TSS concentration and the location of the PSVD curve, with higher TSS concentrations corresponding to higher settling velocities. It was demonstrated that the dynamics of the wastewater characteristics under dry, wet, and snowmelt weather conditions influence grit settling characteristics.

Detection of Erionite and Other Zeolite Fibers in Soil by the Fluidized Bed Preparation Methodology.

Erionite is a zeolite mineral that can occur as fibrous particles in soil. Inhalation exposure to erionite fibers may result in increased risk of diseases, such as mesothelioma. Low level detection of mineral fibers in soils has traditionally been accomplished using polarized light microscopy (PLM) methods to analyze bulk samples providing detection limits of around 0.25% by weight. This detection level may not be sufficiently low enough for protection of human health and is subject to large variability between laboratories. The fluidized bed asbestos segregator (FBAS) soil preparation method uses air elutriation to separate mineral fibers, such as erionite, from soil particles with higher aerodynamic diameter and deposits those mineral fibers onto filters that can be quantitatively analyzed by microscopic techniques, such as transmission electron microscopy (TEM). In this study, performance evaluation (PE) standards of erionite in soil with nominal concentrations ranging from 0.1% to 0.0001% by weight were prepared using the FBAS soil preparation method and the resulting filters were analyzed by TEM. The analytical results of this study illustrate a linear relationship between the nominal concentration of erionite (as % by weight) in the PE standard and the concentration estimated by TEM analysis expressed as erionite structures per gram of test material (s/g). A method detection limit of 0.003% by weight was achieved, which is approximately 100 times lower than typical detection limits for soils by PLM. The FBAS soil preparation method was also used to evaluate authentic field soil samples to better estimate the concentrations of erionite in soils on a weight percent basis. This study demonstrates the FBAS preparation method, which has already been shown to reliably detect low levels of asbestos in soil, can also be used to quantify low levels of erionite in soil.

Microplastics elutriation system: Part B: Insight of the next generation.

Elutriation is an efficient process for extracting microplastics. The development of a numerical model has shown the need for optimizing aspects of the design of the actual elutriation protocol as well as the dimensioning of the column to increase its efficiency. The study aims to propose new dimensioning data and protocol elements for designing an efficient column. Using a numerical model, the filling velocity was calculated as a function of the size and the density of the particles to prevent sand suspension. The sieving protocol was adapted to increase the density limit for the extraction of plastic particles from 1460 to >1800 kg·m-3. The durations of the elutriation and the column height were calculated to improve the control of the particle suspension. These results contribute to the development of the next generation of elutriation system and will accelerate the study of plasticome in the context of sandy sediments.

Microplastic in beach sediments of the Isle of Rügen (Baltic Sea) - Implementing a novel glass elutriation column.

To extent the understanding on microplastics in the marine environment we performed a case study at four beaches on the Isle of Rügen considering abundance and spatial distribution of microplastics in beach sediments. For the analysis, density separation via a glass elutriation column was implemented. In advance, efficiencies were tested for two polymers, being not buoyant in water. Recovery rates of 80% for PET and 72% for PVC particles in sandy samples were achieved. A median abundance of 88.10 (Q1=55.01/Q3=114.72) microplastic particles per kg dry sediment or 2862.56 (Q1=1787.34/Q3=3727.28) particles per m2 was found at the beaches on Rügen. Fibers were more abundant than fragments at all beaches. In this study, no statistically significant differences but only tendencies were determined between the beaches with different exposition and anthropogenic activity as well as for distribution patterns which showed that microplastic fragments accumulate in topographic depressions, similar to macrolitter items.

Effectiveness of piggery waste treatment using microbial fuel cells coupled with elutriated-phased acid fermentation.

The present study evaluates the feasibility of increased power generation in microbial fuel cells (MFCs) coupled with acid elutriation fermentation. Raw piggery waste (RPW) and acid elutriation effluents (AEE) of piggery waste were used to generate bioelectricity in single-chambered air-cathode MFCs. RPW-fed MFCs exhibited stable performance after 12-days of operation, generating 540mV of open circuit voltage (OCV). RPW fed-MFCs displayed peak potential and maximal power density (PDmax) of 0.364V and 192mW/m2 with 980Ω external resistance (Rext), respectively. AEE-fed MFCs documented 818mV of maximum OCV. Furthermore, the peak potential and PDmax of 0.329V and 1553mW/m2 were generated with 100Ω Rext, respectively. RPW and AEE-fed MFCs exhibited 84% and 93% substrate removal efficiency, respectively. These findings suggest that a two-stage process including acid elutriation reactor asa pre-fermentation and MFCs greatly enhances substrate removal and electricity generation from piggery waste.

Microplastics elutriation system. Part A: Numerical modeling.

The elutriation process has shown its efficiency to extract microplastics from sand and began to spread in the scientific community. This extraction technic requires knowing with accuracy the extraction velocities of particles. This study aims to test whether numerical modeling could help to calculate these velocities. From hydrodynamic equations, a numerical model has been developed and the outputs are compared to experimental extraction data. The results show, for the calculated velocities, the experimental plastic extraction yields will be higher than 90% for <10% of sand contamination. The model also allows determining that, with the actual protocol, the maximum plastic density which can be extracted is about 1450kg·m-3 whereas the detrimental resuspension, which may occur during the column filling step, is highlighted. From model calculations, it arises that changes in the column dimensioning and the protocol operations need to be considered.

Ribosome profiling the cell cycle: lessons and challenges.

Understanding the causes and consequences of dynamic changes in the abundance and activity of cellular components during cell division is what most cell cycle studies are about. Here we focus on control of gene expression in the cell cycle at the level of translation. The advent of deep sequencing methodologies led to technologies that quantify the levels of all mRNAs that are bound by ribosomes and engaged in translation in the cell (Ingolia et al. Science 324:218-223, 2009). This approach has been applied recently to synchronous cell populations to find transcripts under translational control at different cell cycle phases (Blank et al. EMBO J 36:487-502, 2017; Stumpf et al. Mol Cell 52:574-582, 2013; Tanenbaum et al. Elife 4:e07957, 2015). These studies revealed new biology, but they also have limitations, pointing to challenges that need to be addressed in the future.

Selection of G1 Phase Yeast Cells for Synchronous Meiosis and Sporulation.

Centrifugal elutriation is a procedure that allows the fractionation of cell populations based upon their size and shape. This allows cells in distinct cell cycle stages can be captured from an asynchronous population. The technique is particularly helpful when performing an experiment to monitor the progression of cells through the cell cycle or meiosis. Yeast sporulation like gametogenesis in other eukaryotes initiates from the G1 phase of the cell cycle. Conveniently, S. cerevisiae arrest in G1 phase when starved for nutrients and so withdrawal of nitrogen and glucose allows cells to abandon vegetative growth in G1 phase before initiating the sporulation program. This simple starvation protocol yields a partial synchronization that has been used extensively in studies of progression through meiosis and sporulation. By using centrifugal elutriation it is possible to isolate a homogeneous population of G1 phase cells and induce them to sporulate synchronously, which is beneficial for investigating progression through meiosis and sporulation. An additionally benefit of this protocol is that cell populations can be isolated based upon size and both large and small cell populations can be tested for progression through meiosis and sporulation. Here we present a protocol for purification of G1 phase diploid cells for examining synchronous progression through meiosis and sporulation.

Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells.

BACKGROUND: Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. METHODS: Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products. RESULTS: When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells. CONCLUSIONS: Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.

Synchronization of Yeast.

The budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are amongst the simplest and most powerful model systems for studying the genetics of cell cycle control. Because yeast grows very rapidly in a simple and economical media, large numbers of cells can easily be obtained for genetic, molecular, and biochemical studies of the cell cycle. The use of synchronized cultures greatly aids in the ease and interpretation of cell cycle studies. In principle, there are two general methods for obtaining synchronized yeast populations. Block-and-release methods can be used to induce cell cycle synchrony. Alternatively, centrifugal elutriation can be used to select synchronous populations. Because each method has innate advantages and disadvantages, the use of multiple approaches helps in generalizing results. An overview of the most commonly used methods to generate synchronized yeast cultures is presented along with working Notes: a section that includes practical comments, experimental considerations and observations, and hints regarding the pros and cons innate to each approach.