Continuous Culture Research Articles

Analysis of full nitrification performance and optimization of reaction properties using N and O isotope fractionation

Isotopic fractionation properties have been successfully applied to identify the distribution and fate of nitrogen in ecosystems, revealing the dynamic response of N and O elements during nitrogen transport and transformation. However, only a few studies used the dual isotope technology in activated sludge treatment of domestic wastewater and many aspects of the process are unclear. Here, we use the dual isotope techniques to increase the understanding of the substrates required for nitrification reactions, nitrification performance, and process operation. Mixed sludge was successfully enriched with nitrifying bacteria in a continuous culture, and three dissolved oxygen (DO; 0.2–0.4, 3–4, and 7–8 mg/L) and three temperature levels (18 ± 1, 25 ± 1, and 33±1 °C) were tested for efficiency of nitrate nitrogen accumulation. Both δ15NNO3 and δ18ONO3 showed a gradual increase with an increase in DO or temperature, the increase in DO slowed down the fractionation effect of isotopes, and the increase in temperature reduced the variability in N and O utilization. The slope of δ15NNO3:δ18ONO3 gradually approached 1 with the increase in DO (<7 mg/L) or in temperature, and the optimal range of DO and temperature were accurately judged to strengthen the denitrification performance of nitrifying bacteria. δ18OH2O was successfully taken up to form NO2−-N and NO3−-N with 74 and 91% replacement rates, respectively, indicating that DO and H2O jointly completed the formation of nitrate nitrogen during the long nitrification process. In summary, the in situ dual isotope technology can help optimize the influence of environmental factors on nitrification performance to guide the long-term stable operation of nitrification reactions in sludge treatment and provide a reliable basis for complex activated sludge nitrification systems.

Production of poly‐gamma‐glutamic acid and spore formation in Bacillus velezensis 83 are affected by acidic growing conditions and glucose uptake rate

AbstractBACKGROUNDBacillus velezensis 83 is a biocontrol agent and plant growth promoting bacteria that produces antifungal compounds (surfactin and bacilomycin D), plant growth stimulation metabolites such as acetoin and butanediol and the polymer poly‐gamma‐glutamic acid (γ‐PGA), which is essential for bacterial plant colonization. Bacillus sporulation is an important trait in order to obtain long shelf‐life formulations for biocontrol purposes. γ‐PGA production and sporulation are closed‐related processes controlled by glucose availability and modulated by quorum sensing. High spore production (and its recovery) in B. velezensis 83 cultures is limited by the high viscosity oxygen‐limited broths resulting from γ‐PGA accumulation. This work studied the influence of acidic growing conditions and glucose specific uptake rate on growth, quorum sensing‐related metabolites production and sporulation of B. velezensis 83.RESULTSIn batch cultures developed at pH = 5, no bacterial sporulation was detected and quorum sensing related metabolites as γ‐PGA (not detected) and surfactin and bacillomycin were significantly decreased. Instead, acetoin and butanediol were the main products. In contrast, in continuous cultures developed at pH = 5, spores were detected at D = 0.06 h−1. The results indicate that sporulation at pH = 5 is not inhibited by affectations on quorum sensing signals but could be due to a low availability/transport of energy sources (i.e., glucose or acetoin/butanediol) or an affectation on Rap‐Phr system.CONCLUSIONThe findings in this study allow to establish a two‐stage process for Bacillus velezensis 83 spore production: bacterial growth at pH = 5 and sporulation at pH = 6.8. © 2023 Society of Chemical Industry (SCI).

Features of SARS-CoV-2 Replication in Various Types of Reptilian and Fish Cell Cultures.

SARS-CoV-2 can enter the environment from the feces of COVID-19 patients and virus carriers through untreated sewage. The virus has shown the ability to adapt to a wide range of hosts, so the question of the possible involvement of aquafauna and animals of coastal ecosystems in maintaining its circulation remains open. the aim of this work was to study the tropism of SARS-CoV-2 for cells of freshwater fish and reptiles, including those associated with aquatic and coastal ecosystems, and the effect of ambient temperature on this process. In a continuous cell culture FHM (fathead minnow) and diploid fibroblasts CGIB (silver carp), SARS-CoV-2 replication was not maintained at either 25 °C or 29 °C. At 29 °C, the continuous cell culture TH-1 (eastern box turtle) showed high susceptibility to SARS-CoV-2, comparable to Vero E6 (development of virus-induced cytopathic effect (CPE) and an infectious titer of 7.5 ± 0.17 log10 TCID50/mL on day 3 after infection), and primary fibroblasts CNI (Nile crocodile embryo) showed moderate susceptibility (no CPE, infectious titer 4.52 ± 0.14 log10 TCID50/mL on day 5 after infection). At 25 °C, SARS-CoV-2 infection did not develop in TH-1 and CNI. our results show the ability of SARS-CoV-2 to effectively replicate without adaptation in the cells of certain reptile species when the ambient temperature rises.

Effects of chicken manure extract on the directed cultivation of bio-bait Chlorella vulgaris-rotifer (Brachionus plicatilis) and their fatty acid content

Chicken manure is rich in various nutrients, and it is used to improve the nutritional status of microalgae, which subsequently affects the production and nutrition of rotifers. The investigation of the effect of chicken manure on Chlorella and rotifer cultivation is therefore critical for practical production. Here, to investigate the effects of chicken manure extract (CME) on the continuous culture of the biological bait “Chlorella-rotifer”, different concentrations of CME (Groups 1–5: 0, 5, 10, 15, and 20 mL·L−1, respectively) were added to the culture medium for cultivating Chlorella vulgaris, assessed the growth of C. vulgaris, and the optimal amount of CME for culturing Chlorella was determined; and the effects of the cultured Chlorella as food for rotifer cultivation and fatty acid composition of Chlorella and rotifers were analyzed. In 12 days of continuous culture, no significant difference in Chlorella population density was noted among G3, G4, and G5 (P > 0.05). Following completion of the culturing period, the specific growth rate of Chlorella in G3 (0.14 ± 0.01) and G5 (0.14 ± 0.01) showed no significant difference (P > 0.05), and chlorophyll a (Chl a) content in G3 (3.79 ± 0.06 mg·mL−1) was significantly higher than that in G5 (3.30 ± 0.12 mg·mL−1) (P < 0.05). Hence, we chose G3-cultured Chlorella for rotifer cultivation. In the 120-h rotifer cultivation period, the longevity of G3-bred rotifers (G-CME) (15.06 ± 0.82 d) was significantly more than that of the control group (G-BG11) (13.13 ± 1.27 d) (P < 0.05), and the offspring number of rotifer in G-CME (24.75 ± 3.45) was significantly higher than that in G-BG11 (17.75 ± 3.06) (P < 0.05). Rotifer population density and fecundity were significantly improved by G-CME than by G-BG11. Regarding fatty acid composition, the principal component analysis showed that Chlorella cultured by CME (CME-C) and BG11 (BG11-C) and rotifers that used them as diet (CME-R and BG11-R, respectively) exhibited a variation in principal components, with LC-PUFAs (12.66% ± 0.42%) and n-6 LC-PUFAs (3.99% ± 0.17%) being significantly higher in CME-R than in BG11-R (LC-PUFAs: 9.76% ± 0.81%, n-6 LC-PUFAs: 2.17% ± 0.32%) (P < 0.01). In conclusion, CME can promote the growth and alter the fatty acid composition of Chlorella, thus indirectly improving the growth condition and fatty acid content of rotifers; the optimal amount of CME for the continuous cultivation of the bio-bait “Chlorella-rotifer” was 10 mL·L−1.

KITSCH, AS AN AESTHETIC CATEGORY IN POSTMODERN ART

This essay aims to bring the artistic scale of kitsch3 that expands from the common to the interdisciplinary, even to hyper-realism, up for discussion by analyzing the kitsch phenomenon, which may be positioned as the aesthetic equivalent of the productions of the continuous culture in the postmodern environment. Yet today’s art, which is confronted with a kind of zeitgeist that has spread to all styles from easily consumable to elitist art, is under the influence of a perception management that dominates almost all daily reality with the guise of cultural management. The aesthetics defined by Baumgarten in 18th century. After that it reshapes according to spirit of time, as late-capitalist order, which exhausted modernism and connected the world, took for granted intricate identity that contains both deep-rooted tradition and contemporary. Kitsch, which classified as banal by Greenberg in 1960s, was tried to be excluded from field of art through Baudrillard's rating of bad, worse, kitsch in 1980s, but the style that Nerdrum glorified with definition of new old masters shifted the discussion from matter of taste to intellectual concern’s field, thus question of quality has become invisible. Kitsch, which has been released in increasingly hybridizing Contemporary Art environment, has spread to many cultural and artistic fields, and today it has almost turned into an aesthetic category.

Model-based control system design to manage process parameters in mammalian cell culture for biopharmaceutical manufacturing.

To enhance the robustness and flexibility of biopharmaceutical manufacturing, a paradigm shift toward methods of continuous processing, such as perfusion, and fundamental technologies for high-throughput process development are being actively investigated. The continuous upstream process must establish an advanced control strategy to ensure a "State of Control" before operation. Specifically, feedforward and feedback control must address the complex fluctuations that occur during the culture process and maintain critical process parameters in appropriate states. However, control system design for industry-standard mammalian cell culture processes is still often performed in a laborious trial-and-error manner. This paper provides a novel control approach in which controller specifications to obtain desired control characteristics can be determined systematically by combining a culture model with control theory. In the proposed scheme, control conditions, such as PID parameters, can be specified mechanistically based on process understanding and control requirements without qualitative decision making or specific preliminary experiments. The effectiveness of the model-based control algorithm was verified by control simulations assuming perfusion Chinese hamster ovary culture. As a tool to assist in the development of control strategies, this study will reduce the high operational workload that is a serious problem in continuous culture and facilitate the digitalization of bioprocesses.

426 Forages Containing Plant Secondary Compounds Can Improve Digestibility, Rumen Ammonia and Rumen Metabolites of Pasture: an in Vitro Continuous Culture Case Study

Abstract Forages containing plant secondary metabolites (PSM) have been investigated for use in reducing nitrogen waste in feedlot and dairy cattle diets but use as supplements to grazing beef cattle to capture the positive effects of PSM is unclear. Here we investigate potential improvement in rumen digestibility and metabolome, rumen ammonia production and flow from feed protein due to PSM bioactivity in forages used to supplement pasture grass using in vitro methods. Can PSM-containing forages offer a practical way to improve range nutrition and provide delivery of phytochemical benefits? Phytochemical- containing forages birdsfoot trefoil (BFT), sainfoin (SAN), small burnet (SMB) and cicer milkvetch (CMV) supplemented grass pasture diets (200 g/kg of DM). In vitro true dry matter digestibility was determined using 2-stage in vitro methods. Randomly assigned diets of PAS supplemented with one of the PSM-containing forage diets (PAS+X) or not supplemented (PAS) were randomly applied to in vitro dual flow continuous culture (CC) vessels inoculated with grazing beef cow rumen fluid as a metabolic host. For 2-stage digestibility, SMB digestibility exceeded that of CMV, followed by BFT and SAN. The grass diet (PAS) was the least digestible (Table 1). Saponin was detected in CMV samples, which is a new finding. For CC, compared with PAS, minimum culture pH for PAS+CMV was highest (P &amp;lt; 0.02) but no differences were observed in pH between PAS, and PAS+BFT and PAS+SAN. Compared with PAS, the molar proportion of the fatty acid propionate increased with PAS+CMV(P = 0.03) in contrast to the acetate- to- propionate ratio found highest for PAS and decreased for all diets with supplementation of PSM forages. Concentration of rumen NH3–N flow to rumen fluid per gram of protein intake was lower (P &amp;lt; 0.01) with PAS+CMV and PAS+SMB, compared with grass only. Feeding CMV and SMB to grazing cattle may improve nutrient utilization, metabolome, and environmental sustainability of beef cattle production by improving diet digestibility and reducing ammonia waste.

PSVI-30 Effects of Different Cobalt Sources on Ruminal Fermentation of Lactating Dairy Cow Diets in a Dual-Flow Continuous Culture System

Abstract The objective of this study was to evaluate the effects of different sources of cobalt in a lactating dairy cow diet on in vitro ruminal fermentation. All diets provided the same amount of supplemental Co (1 mg per kg of diet DM) included in a premix with a targeted inclusion level of 2.5% of diet DM. Treatments were cobalt carbonate (CON), and three alternative Co sources; alternative source 1 (AS1), alternative source 2 (AS2), and alternative source 3 (AS3). Eight dual-flow continuous culture fermenters were used in a replicated 4 × 4 Latin square. Experimental periods were 10 d (7 d adaptation and 3 d sample collection). Composite samples of daily effluents were collected and analyzed for NH3-N, lactate, and VFA concentration as well as for nutrient digestibility. Samples of ruminal content were collected from each fermenter at 0, 1, 2, 4, 6, and 8 h after feeding to determine lactate, NH3-N, and pH kinetics. All data were analyzed using PROC MIXED of SAS and the repeated variable of time was included for pH, lactate, and NH3-N. There were neither treatment effects nor interactions between treatment and time for pH, NH3-N, and lactate concentrations, indicating a similar Co kinetics across treatments. With regards to lactate pool, we did not observe treatment effects for L-lactate; however, we observed treatment effects for D-lactate and total-lactate as seen in Table 1. Analyses of total and individual VFA, N utilization, and nutrient degradation are still undergoing, hence, it is possible that Co sources have effects on ruminal fermentation.

PSII-16 Comparison of Two Methods of Inoculation of Continuous Culture Fermentors Fed Different Diets

Abstract This study assessed how methods of inoculating continuous culture fermentors with rumen particulate digesta affected in vitro fermentation. A 4-unit, single-flow fermentor system was used in a 4x4 Latin Square with treatments arranged in a 2x2 factorial design. Dietary treatments were 1) a corn-silage-based total mixed ration (TMR) diet; and 2) an orchardgrass (Dactylis glomerata L.) and ground corn-based (ORG) diet balanced to support similar levels of milk production. Within each diet, two inoculation methods were used 1) direct addition of particulate digesta (32 g) into the fermentor vessel (-S); or 2) blending an equivalent quantity of particulate digesta with 0.5 L of rumen liquid for 2 minutes, straining through two layers of cheesecloth, and adding only the strained liquid to the vessel (-B). All fermentors were inoculated with identical total quantities of rumen liquid (1.5 L). This resulted in four treatments, ORG-S, ORG-B, TMR-S, and TMR-B, which were randomly assigned to fermentors with 7 d of treatment adaptation and 3 d of sample collection. Effluent samples (24-h composite) were collected on d 8-10, composited by fermentor within period, and analyzed for volatile fatty acids (VFA), and organic matter (OM) and crude protein (CP) for determination of nutrient digestibilities. Data were analyzed using a mixed model with period and treatment as fixed effects, and fermentor as a random effect. Preplanned contrasts were used to compare the effect of inoculation method within each diet, and the interaction between inoculation method and diet. Apparent OM digestibility was 16% greater in ORG-S compared with ORG-B (P &amp;lt; 0.01), whereas no effect of inoculation method was observed in the TMR diet (P = 0.36), which resulted in an interaction of diet and inoculation method (P &amp;lt; 0.01). Concentration of total VFA was greater with direct addition of particulate digesta in both diets, though there was a significant interaction of diet and inoculation method (P &amp;lt; 0.01) due to a greater increase in VFA in the ORG diet (34%; P &amp;lt; 0.01) compared with TMR (10%; P = 0.01). Crude protein digestibility tended (P = 0.06) to be greater in ORG-S vs. ORG-B, whereas inoculation method did not affect CP digestibility in the TMR diet (P = 0.58). Inoculation method affected methane production (mmol/d) only in the ORG diet; total methane production was 58% greater in ORG-S compared with ORG-B (P &amp;lt; 0.01), whereas methane production was not different in TMR-S vs. TMR-B (P = 0.76), leading again to an interaction of diet and inoculation method (P = 0.04). In general, adding particulate digesta directly to fermentor vessels increased feed degradation and methane production in the ORG diet, but had a lesser effect in the TMR diet.

Characterization and identification of primarily cultured astrocytes and microglia

The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7 % of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.

Microcystin aids in cold temperature acclimation: Differences between a toxic Microcystis wildtype and non-toxic mutant

For Microcystis aeruginosa PCC 7806, temperature decreases from 26 °C to 19 °C double the microcystin quota per cell during growth in continuous culture. Here we tested whether this increase in microcystin provided M. aeruginosa PCC 7806 with a fitness advantage during colder-temperature growth by comparing cell concentration, cellular physiology, reactive oxygen species damage, and the transcriptomics-inferred metabolism to a non-toxigenic mutant strain M. aeruginosa PCC 7806 ΔmcyB. Photo-physiological data combined with transcriptomic data revealed metabolic changes in the mutant strain during growth at 19 °C, which included increased electron sinks and non-photochemical quenching. Increased gene expression was observed for a glutathione-dependent peroxiredoxin during cold treatment, suggesting compensatory mechanisms to defend against reactive oxygen species are employed in the absence of microcystin in the mutant. Our observations highlight the potential selective advantages of a longer-term defensive strategy in management of oxidative stress (i.e., making microcystin) vs the shorter-term proactive strategy of producing cellular components to actively dissipate or degrade oxidative stress agents.

Effect of calcium-independent phospholipase A2 on the expression of glycerol 3-phosphate dehydrogenase in human non-alcoholic fatty liver disease cells

Objective: To explore the effect of calcium-independent phospholipase A2 (iPLA2) on the expression of mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) in human non-alcoholic fatty liver disease cells. Methods: Oleic acid was used to construct a non-alcoholic fatty liver disease cell model by inducing lipid deposition in THLE-2 cells in vitro. Simultaneously, intracellular triglyceride content, iPLA2 expression levels, and mGPDH levels were determined at various induction times (0, 24, 48, and 72 h) using a triglyceride assay kit, quantitative RT-PCR, and western blotting. The model cells were treated with bromelenol lactone, an iPLA2 inhibitor, and N-acetylcysteine, a ROS inhibitor, respectively. Following continuous culture for 24 and 48 hours, the cells were harvested, and the mRNA and protein expression levels of mGPDH were measured. Statistical analysis was performed using the t-test, one-way analysis of variance, and linear correlation. Results: The intracellular triglyceride content gradually increased (P < 0.01), the mGPDH mRNA and protein expression decreased (P < 0.01), and the iPLA2 mRNA and protein expression increased (P < 0.01) in THLE-2 cells with the prolonging time effect of oleic acid therapy. In addition, the mGPDH mRNA expression level was negatively correlated with the iPLA2 mRNA level (r = -0.878, P = 0.002). The expression levels of mGPDH mRNA and protein in the iPLA2 inhibitor group and ROS inhibitor group were increased compared with the model control group (P < 0.01). The expression of mGPDH mRNA was increased at 24 h compared with 48 h in the iPLA2 inhibitor group (P < 0.01). The expression of mGPDH mRNA was gradually increased in the ROS inhibitor group with the prolongation of inhibitor action time (P < 0.01). Compared with the two inhibitor groups, the increase in mGPDH mRNA was significantly higher in the ROS inhibitor group than that in the iPLA2 inhibitor group, and the difference was statistically significant (P < 0.01). Conclusion: iPLA2 can inhibit the expression of mGPDH in non-alcoholic fatty liver cells to a certain extent.

Continuous culture of anaerobic fungi enables growth and metabolic flux tuning without use of genetic tools

Anaerobic gut fungi (AGF) have potential to valorize lignocellulosic biomass owing to their diverse repertoire of carbohydrate-active enzymes (CAZymes). However, AGF metabolism is poorly understood, and no stable genetic tools are available to manipulate growth and metabolic flux to enhance production of specific targets, e.g., cells, CAZymes, or metabolites. Herein, a cost-effective, Arduino-based, continuous-flow anaerobic bioreactor with online optical density control is presented to probe metabolism and predictably tune fluxes in Caecomyces churrovis. Varying the C. churrovis turbidostat setpoint titer reliably controlled growth rate (from 0.04 to 0.20 h−1), metabolic flux, and production rates of acetate, formate, lactate, and ethanol. Bioreactor setpoints to maximize production of each product were identified, and all continuous production rates significantly exceed batch rates. Formate spike-ins increased lactate flux and decreased acetate, ethanol, and formate fluxes. The bioreactor and turbidostat culture schemes demonstrated here offer tools to tailor AGF fermentations to application-specific hydrolysate product profiles.

Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79g/L and 6.8u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025h-1 , DCW increased to 88.39g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80mL/h, a recombinant uricase activity was 4.13, 7.2, and 0u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04u/mL/h (0.0017mg/mL/h) to 0.18u/mL/h (0.0078mg/mL/h).

Oxygen respiration and polysaccharide degradation by a sulfate-reducing acidobacterium

Sulfate-reducing microorganisms represent a globally important link between the sulfur and carbon cycles. Recent metagenomic surveys expanded the diversity of microorganisms putatively involved in sulfate reduction underscoring our incomplete understanding of this functional guild. Here, we use genome-centric metatranscriptomics to study the energy metabolism of Acidobacteriota that carry genes for dissimilation of sulfur compounds in a long-term continuous culture running under alternating anoxic and oxic conditions. Differential gene expression analysis reveals the unique metabolic flexibility of a pectin-degrading acidobacterium to switch from sulfate to oxygen reduction when shifting from anoxic to oxic conditions. The combination of facultative anaerobiosis and polysaccharide degradation expands the metabolic versatility among sulfate-reducing microorganisms. Our results highlight that sulfate reduction and aerobic respiration are not mutually exclusive in the same organism, sulfate reducers can mineralize organic polymers, and anaerobic mineralization of complex organic matter is not necessarily a multi-step process involving different microbial guilds but can be bypassed by a single microbial species.

STRATEGIC APPROPRIATION ON HUSTLE CULTURE AS POSITIVE TREND

Hustle culture is a cultural phenomenon that was born outside and was adopted in Indonesia as a continuous culture of workaholism. Hustle also plays an important role at work, because hustle is a new movement for workers to commit to their company. Hustle can also be categorized as good behavior. Because workers give their full role and work optimall y. In Indonesia, this culture continues to develop, especially the millennial generation of teenagers. It is easier for them to work hard, even to generate fantasy income. They adopt workaholic behavior to become financially successful. There are many rejections and disagreements between researchers regarding this Hustle culture. research data is a mix of methods by combining qualitative research and literature review which contains searches of relevant concepts and theories using research objectives. By involving a combination of fieldwork, social media observations, and interviews. The purpose of this literature and field study is to describe hustle culture in Indonesia as a positive trend for teenagers in Indonesia.

Frequent, infinitesimal bottlenecks maximize the rate of microbial adaptation.

Serial passaging is a fundamental technique in experimental evolution. The choice of bottleneck severity and frequency poses a dilemma: longer growth periods allow beneficial mutants to arise and grow over more generations, but simultaneously necessitate more severe bottlenecks with a higher risk of those same mutations being lost. Short growth periods require less severe bottlenecks, but come at the cost of less time between transfers for beneficial mutations to establish. The standard laboratory protocol of 24-h growth cycles with severe bottlenecking has logistical advantages for the experimenter but limited theoretical justification. Here we demonstrate that contrary to standard practice, the rate of adaptive evolution is maximized when bottlenecks are frequent and small, indeed infinitesimally so in the limit of continuous culture. This result derives from revising key assumptions underpinning previous theoretical work, notably changing the metric of optimization from adaptation per serial transfer to per experiment runtime. We also show that adding resource constraints and clonal interference to the model leaves the qualitative results unchanged. Implementing these findings will require liquid-handling robots to perform frequent bottlenecks, or chemostats for continuous culture. Further innovation in and adoption of these technologies has the potential to accelerate the rate of discovery in experimental evolution.

A STUDY ON VARIANCE ANALYSIS AND ITS EFFECT ON PROFITABILITY AT VARSHA MULTI-TECH RAJAJINAGAR

The vital area of analysis of variance and its substantial effects on corporate profitability are explored in depth in the current research. One of the most important technique for determining the discrepancy between expected and actual results, providing insight into the efficacy and efficiency of management choices. This study methodically examines the many facets of variations, including both positive and negative departures from expected performance. This study reveals the complex causal links that explain variations in profitability by analysing the complicated interplay among cost, revenue, and operational variances. The paper explains each of the ways that variance analysis effects strategic choice and resource allocation through a thorough examination of pertinent literature and direct observation of various sectors. The results highlight the crucial role variance analysis plays in spotting operational inefficiencies, maximising resource use, and promoting a continuous improvement culture inside organisations. In the end, this study adds to a comprehensive knowledge of how variance analysis functions as a compass for guiding companies towards increased profitability in the face of dynamic and difficult market conditions.

Didjeridu acoustics: A review

The didjeridu is the principal musical instrument of the world’s oldest continuous culture and its sound is an acoustic icon of Australia. This lip-valve instrument usually produces a single, sustained, low note. The musical interest comes from rhythmic changes in timbre, including those associated with ‘circular breathing’, wherein exhalation from lungs to instrument with raised velum is alternated with expulsion of air from the inflated cheeks while air is quickly inhaled through the nose, with the velum lowered. This paper reviews research from our lab. Measurements of the acoustic impedance spectrum in the mouth during playing show that impedance peaks produce antiformants or minima in the output sound spectral envelope. The instrument’s slight flare and irregular bore geometry produce resonances that do not fall in harmonic ratios. Instruments judged good by players have particularly weak resonances in the formant frequency region so that the vocal tract impedance peaks can dominate the sound spectrum more easily. In one technique, players phonate at frequencies above that of the instrument, producing complicated heterodyne components. Lips are low Q oscillators and, as in other lip-valve instruments, the energetics of lip oscillation depends on the phase difference between the lips’ longitudinal and lateral motion.

City and Brain Analogy: a Sample for Conservative Versus Adaptive Phenotypical Vision of a Genotypical Heritage

Historical urban settlements and traditional structures are significant indicators and meaningful documents of past ways of life. From a contextual perspective, the preservation of these values and the revitalization of architectural heritage are like sustaining through the conservation of the integrated and continuous culture tied to the past just like a human brain does. The present study encompasses all dimensions of a strategic model to conservation versus adaptation developed for one of the historical building types (Vienna Gasometers), which holds substantial historical values. It involves addressing functions, spatial organization, the manner of interaction with historical building remnants, old-new compatibility, form, structure, and material usage, as well as conducting architectural evaluations. In this study, first; following the architectural assessment of each monumental structure, their spatial, formal, functional, and structural relationships with each other are examined to grow these essences and values for a new vision of the city. Second, the advantages and disadvantages of the conservation approach are critiqued in comparison to adaptive renovation of the city heritages. As an illustrative example, the Simmering region and Gasometer structures located in the outer parts of Austria which is historically the most iconic heritage have been chosen. The preservation of the physical characteristics of Gasometer monumental structures while renovation of the functions of the space within this framework reflect not only Europe's industrial, historical, and social evolution but also hold significance in terms of safeguarding socio-economic and cultural values. Furthermore, they contribute to shaping contemporary notions of conservation while adapting them to the vision of the new era.