An ultra-sensitive and easy-to-use multiplexed single-cell proteomic analysis

Proteins analysis from an average cell population often overlooks the cellular heterogeneity of expressed effector molecules, and knowledge about the regulations of key biological processes may remain obscure. Therefore, the necessity of single-cell proteomics (SCP) technologies arises. Without microfluidic chip, expensive ultrasonic equipment, or reformed liquid chromatogram (LC) system, we established an Ultra-sensitive and Easy-to-use multiplexed Single-Cell Proteomic workflow (UE-SCP). Specifically, the flexible sorting system ensured outstanding cell activity, high accuracy, remarkable efficiency, and robustness during single-cell isolation. Multiplex isobaric labeling realized the high-throughput analysis in trapped ion mobility spectrometry coupled with quadrupole time-of-flight mass spectrometry (timsTOF MS). Using this pipeline, we achieved single-cell protein quantities to a depth of over 2,000 protein groups in two human cell lines, Hela and HEK-293T. A small batch experiment can identify and quantify more than 3200 protein groups in 32 single cells, while a large batch experiment can identify and quantify about 4000 protein groups in 96 single cells. All the 128 single cells from different cell lines could been unsupervised clustered based on their proteomes. After the integration of data quality control, data cleaning, and data analysis, we are confident that our UE-SCP platform will be easy-to-marketing popularization and will promote biological applications of single-cell proteomics.

High-Solid Loading Enzymatic Hydrolysis of Waste Office Paper for Poly-3-Hydroxybutyrate Production Through Simultaneous Saccharification and Fermentation

Waste paper holds great potential as a substrate for the microbial production of bioplastic (Poly-3-hydroxybutyrate (PHB)). This study aimed to produce PHB by utilizing office paper as a substrate using Cupriavidus necator through batch and fed-batch simultaneous saccharification and fermentation (SSF) approach. For the batch experiment, different loadings of shredded office paper (3, 5 and 10%) with two different pretreatments H2O2 (OPH) and H2O2 and Triton X-100 (OPTH) were carried out. For the fed-batch experiment, paper loading started with 3% and two more additions were made at 36 and 84 h. Both experiments were conducted at 30°C, 200 rpm and pH 7 using 55.5 FPU/g of cellulase and 37.5 CBU/g of β-glucosidase with a fixed amount of nitrogen source. High PHB yield was observed with OPH in all loadings, though the OPHT showed a better hydrolysis. Maximum PHB yield (4.27 g/L) was achieved with 10% OP at six days of fermentation in batch SSF. Whereas, maximum PHB yield (4.19 g/L) was obtained within a shorter time (66 h) in the fed-batch OPH paper. The extracted PHB showed well-matched characteristic features to the standard PHB. Finally, this study proves the feasibility of employing SSF process for PHB production using waste paper as an alternative approach to overcome the shortcoming of the separate hydrolysis and fermentation (SHF) process.

The impact of crude glycerol from biodiesel production and its trace element content on biomethane production in a batch experiment: modelling as a step towards impartial routine comparison of results

In this study, crude glycerol from the biodiesel industry was tested as a co-substrate in biogas production. To investigate the influence of crude glycerol and the underlying trace element (TE) content on the efficiency of biomethane production, a batch experiment using Automatic Methane Potential Test System (AMPTS II) was carried out. The single addition of crude glycerol significantly contributed only to the total content of K (14.4%), Si (17.3%), and P (11.6%), whereas the contributions of other metals were within the range of other substrates. The addition of crude glycerol increased biomethane production, however, its utilization beyond 1% of total volume resulted in prolonged lag phase and final cessation of biomethane production. The negative effects of inorganic salts present in crude glycerol were reflected in progressively diminishing parts of glycerol and methanol being utilized in its anaerobic digestion, posing serious problems for daily routine use. A nonlinear least square regression analysis was performed to fit the Gompertz, Logistic, Transfer, and Richards models to biomethane production. The most suitable model was the Richards model, exhibiting the best fit to the experimental curves for complex substrates. Glycerol fractions remaining after biodiesel production have to be pre-tested for their negative effects on the content of TEs and inorganic salts, lag phase in biogas production, before they are used as co-substrates in biogas production phase.

Mechanisms at Different pH for Stabilization of Arsenic in Mine Tailings Using Steelmaking Slag

The mechanisms at different pH for the stabilization of arsenic (As) in mine tailings (MTs) using steelmaking slag were investigated using laboratory experiments. Two types of steelmaking slag were used in the experiments. Ca-slag has high pH and high calcium oxide content due to its short period of aging. In contrast, Fe-slag is oxidized for a long time and is richer in Fe than in Ca. The As-contaminated MTs were taken from a tailing-storage dam around an abandoned gold mine in Korea. The tailings had an average As concentration of 2225.3 mg/kg. The As-removal batch experiment was performed to investigate the As-removal characteristics of the steelmaking slag. From SEM/EDS analyses after each batch experiment, Ca-As bearing precipitates were broadly found on the surface of Ca-slag particles and the final pH of the solution increased to 12.3. However, for Fe-slag, the As was locally found as forms adsorbed to the surface of Fe and Mn oxides contained in the Fe-slag particles. The final pH of this solution was 8.4. The efficiency of removal of As from water using the Ca-slag was >97% and that with Fe-slag was 79%. This suggests that As ions in solution were removed by Ca-(co-)precipitation (which occurs comprehensively on the Ca-slag surface), or by restrictive adsorption of Fe- and Mn-oxides (on limited parts of the Fe-slag). To determine the efficiency of As-extraction reduction from MTs using steel slags, arsenic-extraction batch experiments with two slags were performed under acidic conditions, simulating the leaching environment formed around a mine tailing storage dam. The As concentration in the extracted solution was decreased by 69.9% (even at pH 2) after the addition of 5% Fe-slag. However, when 5% Ca-slag was added, the As concentration decreased by 42.3% at pH 2. These results suggest that Fe-rich steel slag can be more effective than Ca-rich steel slag as a stabilizer for As in contaminated mine tailings at low pH.