Culture Temperature Research Articles

Rhodotorula paludigena CM33 cultivation process development for high β-carotene single cell protein production

Rhodotorula yeast has potential in terms of biotechnology for the production of various compounds for the environmental, industrial, and medical sectors. It is well known to generate carotenoid pigments that are beneficially distinguished. This investigation aimed to optimize the growth of Rhodotorula paludigena CM33 and its production of β-carotene. Response Surface Methodology was employed to optimize temperature, ammonium sulfate, and ferric chloride concentrations in a 5L-bioreactor batch culture. The optimal conditions of 30 °C, 6.2 g/L of ammonium sulfate, and 75.0 mg/L of ferric chloride provided biomass of 17.71 g/L, a specific growth rate of 0.22 h−1, and β-carotene concentration of 40.29 mg/L. Upon scaling up the cultivation process, the 22L-bioreactor displayed similar growth patterns to those of the 5L-bioreactor. However, the 22L-bioreactor exhibited higher product yields in a shorter time period. The maximum biomass production of 18.96 ± 1.31 g/L and the maximum β-carotene production of 42.81 ± 0.61 mg/L were achieved at 28 h. This scaling-up strategy resulted in improved production efficiency. Additionally, the nutritional value of the cells was evaluated, suggesting their potential as animal feed supplements. R. paludigena CM33 demonstrated a β-carotene single-cell protein content, making it a valuable source for aquaculture and livestock feed. These findings highlight the potential of this yeast strain for large-scale production of β-carotene and single-cell protein, offering nutritional benefits for animal feed.

The impact of growing season on the ethylene biosynthesis and signaling pathways of a heat tolerant tomato during off-vine postharvest ripening

Ethylene initiates and mediates fruit ripening throughout a cascade of signal transductions from ethylene receptors (ETRs) to ethylene response factors (ERFs). High temperatures impair fruit ripening by suppressing ethylene biosynthesis and parts of physiological and biochemical processes. This study made use of the ethylene inhibitor (1-MCP) and exogenous ethylene to investigate the ethylene biosynthesis and signaling pathways of a heat tolerant tomato cultivar, cv. ‘Savior’, during off-vine postharvest ripening during two distinct growing seasons. Mature green ’Savior‘ tomatoes grown in winter (mild temperature) and summer (high temperature) were harvested at and treated with 1-MCP or exogenous ethylene. During off-vine ripening, summer fruit showed a different ethylene biosynthesis profile compared to winter fruit. The application of exogenous ethylene did not accelerate fruit ripening of the heat tolerant cultivar. 1-MCP suppressed ethylene production for a long time, and delayed fruit ripening of winter tomato, while for summer fruit, a limited effect of 1-MCP on inhibiting ethylene biosynthesis and delaying ripening was observed despite the strong suppression of ACC oxidase (ACO) activity. Our work also showed that a 1-MCP treatment reduced gene expression of several ethylene receptors (ETR) but did not have a strong effect on constitutive triple response (CTR), except CTR1, and ethylene-insensitive protein 2 (EIN2). Overall, our results indicate that i) ethylene biosynthesis is differentially regulated in winter as compared to summer fruit by ethylene signaling, ii) a high cultivation temperature impacts ethylene biosynthesis and ethylene sensitivity during fruit ripening despite the fact that this is a heat tolerant cultivar, iii) the expression of most ETRs and CTRs, and especially EIN2 did not respond to the 1-MCP or ethylene treatment.

Optimization of Growth Condition for Aquatic Fungi (Saprolegnia species) Isolated from Economically Important Freshwater Fishes

The present study was aimed to optimize the culture media, temperature, pH and sodium chloride concentration for the growth of isolated and characterized Saprolegnia strains which were isolated from four economically important infected fish specis i.e. Catla catla, Labeo rohita, Cirrhinus mrigala and Cyprinus carpio. Identification and characterization of Saprolegnia species was made by observing colony morphology and asexual reproductive organs under binocular light microscope. The result showed that the Saprolegnia sp. grown well in Sabourd’s Dextrose Agar media at optimum temperature 25oC, neutral pH 7 and 0.01% sodium chloride content. The progress of optimization is helpful to measure the occurrence, distribution, outbreaks and epidemiological survey of aquatic fungi. This susceptibility study of pathogen provides an idea to prevent before the onset/outbreak of infectious diseases and helpful in designing the bio-control agent for the Saprolegnia strains.

Novel Strain of Paenibacillus phyllosphaerae CS-148 for the Direct Hydrolysis of Raw Starch into Glucose: Isolation and Fermentation Optimization.

The conventional process for converting starch to glucose is energy-intensive. To lower the cost of this process, a novel strain of Paenibacillus phyllosphaerae CS-148 was isolated and identified, which could directly hydrolyze raw starch into glucose and accumulate glucose in the fermentation broth. The effects of different organic and inorganic nitrogen sources, the culture temperature, the initial pH, and the agitation speed on the yield of glucose were optimized through the one-factor-at-a-time method. Nine factors were screened by Plackett-Burman design, and three factors (raw corncob starch, yeast extract and (NH4)2SO4) had significant effects on glucose yield. Three significant factors were further optimized using Box-Behnken design. Under the optimized fermentation conditions (raw corncob starch 40.4 g/L, yeast extract 4.27 g/L, (NH4)2SO4 4.39 g/L, KH2PO4 2 g/L, MgSO4`7H2O 2 g/L, FeSO4`7H2O 0.02 g/L, NaCl 2 g/L, KCl 0.5 g/L, inoculums volume 4%, temperature 35 °C, agitation rate 150 rpm, and initial pH 7.0), the maximum glucose yield reached 17.32 ± 0.46 g/L, which is 1.33-fold compared to that by initial fermentation conditions. The maximum conversion rate and glucose productivity were 0.43 ± 0.01 g glucose/g raw corn starch and 0.22 ± 0.01 g/(L·h), respectively. These results implied that P. phyllosphaerae CS-148 could be used in the food industry or fermentation industry at a low cost.

Сolonial–morphological characteristics of dissociatives of the Rhodococcus ruber - 8/4/1 strain

This article provides a study of the molecular, physiological, biochemical and morphological features of R-, S- and M-dissociants of Rhodococcus ruber - 8/4/1. The sequence similarity of the genome fragments of R- and S-dissociant forms encoded by 16S rRNA was demonstrated. MALDI spectra were obtained for the R. ruber-8/4/1 strain and its dissociants. Under standard analysis conditions, the MALDI profiles of R-, S- and M- forms dissociants of the Rhodococcus ruber - 8/4/1 were similar. The determination of the resistance of the strain dissociants to external factors showed that R-forms were more stable under UV irradiation and increased temperature. The S-forms showed stability upon the conversion of acrylic acid nitrile into acrylamide (AA), whereas the M-forms were stable under a decrease in the cultivation temperature, and an increase in the concentration of NaCl in the medium. The S-forms proved to be the most active in the transformation of acrylonitrile into AA.

Effect of Pre-Germination Temperature Regime on Pollen Germination and Fruit Set in Pear, Pyrus bretschneiderilia

Extensive research has been conducted on the effects of pollen activation temperature, cultivation temperature, and ambient temperature on germination rates and fruit sets. Yet, the influence of the temperature (pre-germination temperature) at which the pollen suspension is prepared within the storage container (tank) remains unexplored. In this study, we initially established the optimal time for pollen activation. Subsequently, pollen suspensions at varying temperatures were prepared, and their germination rates were analyzed using microscopy. Pollen suspensions of different temperatures were then sprayed onto Fojianxi pear flowers, and parameters such as flower fruit set rate, inflorescence fruit set rate, fruit longitudinal dimension, fruit transverse dimension, and fruit shape index were evaluated. The findings revealed that the optimal pollen activation duration was 6 h. A pollen suspension temperature of 30 °C resulted in the highest germination rate (72.06 ± 6.87%). However, a suspension temperature of 25 °C achieved the highest flower fruit set rate (57.29 ± 5.58%) and inflorescence fruit set rate (87.50 ± 4.84%). The fruit longitudinal dimension (68.12 ± 3.94 mm), transverse dimension (73.77 ± 4.04 mm), and fruit shape index (108.42 ± 6.02%) were most favorable at a suspension temperature of 15 °C. Our study concludes that the pollen in lower pollen suspension temperatures (15~25 °C) has higher germination energy and have higher stigmatic capacity. Thus, we advocate for a pollen suspension temperature range of 15~25 °C when employing liquid spray pollination techniques with Xuehuali (Pyrus bretschneiderilia) pollen.

Optimizing ErCas12a for efficient gene editing in Arabidopsis thaliana.

The ErCas12a nuclease, also known as MAD7, is part of a CRISPR/Cas system from Eubacterium rectale and distantly related to Cas12a nucleases. As it shares only 31% sequence homology with the commonly used AsCas12a, its intellectual property may not be covered by the granted patent rights for Cas12a nucleases. Thus, ErCas12a became an attractive alternative for practical applications. However, the editing efficiency of ErCas12a is strongly target sequence- and temperature-dependent. Therefore, optimization of the enzyme activity through protein engineering is especially attractive for its application in plants, as they are cultivated at lower temperatures. Based on the knowledge obtained from the optimization of Cas12a nucleases, we opted to improve the gene editing efficiency of ErCas12a by introducing analogous amino acid exchanges. Interestingly, neither of these mutations analogous to those in the enhanced or Ultra versions of AsCas12a resulted in significant editing enhancement of ErCas12a in Arabidopsis thaliana. However, two different mutations, V156R and K172R, in putative alpha helical structures of the enzyme showed a detectable improvement in editing. By combining these two mutations, we obtained an improved ErCas12a (imErCas12a) variant, showing several-fold increase in activity in comparison to the wild-type enzyme in Arabidopsis. This variant yields strong editing efficiencies at 22 °C which could be further increased by raising the cultivation temperature to 28 °C and even enabled editing of formerly inaccessible targets. Additionally, no enhanced off-site activity was detected. Thus, imErCas12a is an economically attractive and efficient alternative to other CRISPR/Cas systems for plant genome engineering.

Establishment of a Single Temperature Incubation Approach for Environmental Monitoring Samples with Focus on Mold Recoveries.

A robust environmental monitoring program is essential to properly estimate and identify microorganisms in cleanrooms, ensuring that microbial contamination remains acceptably low, and a good state of control is maintained in the manufacturing areas. The incubation conditions are important to support optimal microbial recoveries, considering that there is no single culture medium, temperature and incubation time that can recover all microorganisms. Particularly molds are quite sensitive microorganisms, and some species may have very specific nutritional and environmental needs. In this study, a two-phase approach was used to identify a single incubation-temperature approach that could recover most of the cleanroom microbial flora with focus on molds. Phase 1 included a growth promotion study performed in the laboratory using pharmacopeial and in-house strains, comparing different media (SDA and TSA) at single or dual incubation-temperature approaches for 5 or 6 days. Phase 2 was based on an in situ study where sampling was performed in different areas of a pharmaceutical facility, comparing the recoveries at different incubation conditions. In addition, extension studies of phase 1 and phase 2 were performed to get a better understanding of growth requirements for in-house molds. The results show that an incubation on Tryptone Soy Agar (TSA) at 25-30° for 3-4 days was able to recover most tested microorganisms in phase 1 and a large variety of microorganisms in phase 2, indicating that the single incubation-temperature is an optimal approach for the recovery of microorganisms in cleanrooms. Exceptions were noted for one strain of the species Cutibacterium acnes, a microaerophilic bacterium for which anaerobiosis and higher temperatures were needed, and two mold strains (Sistotrema brinkmannii and Stereum hirsutum), indicating that those molds required a specific media (Sabouraud Dextrose Agar, SDA) for their proliferation. The results showed that TSA incubated at single or dual incubation-temperature approach cannot compensate for the absence of SDA for some environmental molds that may be atypical in cleanrooms. Therefore, in addition to TSA, certain monitoring with SDA at e.g., cleanroom entrance points may be beneficial to recover molds with very specific nutritional requirements.

Novel methodologies for host-microbe interactions and microbiome-targeted therapeutics in 3D organotypic skin models

BackgroundFollowing descriptive studies on skin microbiota in health and disease, mechanistic studies on the interplay between skin and microbes are on the rise, for which experimental models are in great demand. Here, we present a novel methodology for microbial colonization of organotypic skin and analysis thereof.ResultsAn inoculation device ensured a standardized application area on the stratum corneum and a homogenous distribution of bacteria, while preventing infection of the basolateral culture medium even during prolonged culture periods for up to 2 weeks at a specific culture temperature and humidity. Hereby, host-microbe interactions and antibiotic interventions could be studied, revealing diverse host responses to various skin-related bacteria and pathogens.ConclusionsOur methodology is easily transferable to a wide variety of organotypic skin or mucosal models and different microbes at every cell culture facility at low costs. We envision that this study will kick-start skin microbiome studies using human organotypic skin cultures, providing a powerful alternative to experimental animal models in pre-clinical research.Dkr_4cuqp92zgpqQmj5SV7Video

Cyanobacterial bioreporter of nitrate bioavailability in aquatic ecosystems

The water eutrophication, resulting from the discharge of industrial and agricultural wastewater, leads to ecological degradation. However, to date, how to assess and manage the risks of water pollution, especially nitrogen pollution, remains a particularly noteworthy issue. Nitrate, the most important nitrogen compound, has become a bottleneck restricting total nitrogen management. The development of bioreporters monitoring nitrate pollution contributes to the estimation of water quality, especially the availability of nutrients. In this study, we obtained 9 bioreporters from 40 cyanobacterial derivatives which were constructed based on different hosts, copy numbers, and sensing elements and evaluated the performance of bioreporters. The results showed that single-celled Synechocystis was more sensitive to nitrate than filamentous Anabaena, that the reporter gene luxABCDE responded faster than sfgfp in most bioreporters, and that relatively medium-copy plasmid improved the performance of sensing elements. Nine bioreporters performed well in bioavailable nitrate detection, of which AD-AS-X and AR-NI-X, activated by nitrate repletion, had the shortest response time (2 h) and the widest response range (20–800 μM), respectively. Moreover, SR-GLN-SG, activated by nitrate deficiency, exhibited the best linear response (R2 = 0.998). After parameter optimization, exponential growth phase bioreporters, culture temperature of 30 °C, sample volume of 200 μL were determined as optimal monitoring conditions. We found that common water contaminants (copper, cadmium, and phosphorus) had no impact on the performance of bioreporters, indicating the stability of bioreporters. Six out of 9 bioreporters, especially the SR-NB-X, were highly effective in detecting the bioavailable nitrate in wastewater sample. This study provides valuable references for developing more cyanobacterial bioreporters and their practical application in nitrate detection.

Optimal temperature for the long-term culture of adult porcine islets for xenotransplantation.

Porcine islet xenotransplantation represents a promising therapy for severe diabetes mellitus. Long-term culture of porcine islets is a crucial challenge to permit the on-demand provision of islets. We aimed to identify the optimal temperature for the long-term culture of adult porcine islets for xenotransplantation. We evaluated the factors potentially influencing successful 28-day culture of islets at 24°C and 37°C, and found that culture at 37°C contributed to the stability of the morphology of the islets, the proliferation of islet cells, and the recovery of endocrine function, indicated by the expression of genes involved in pancreatic development, hormone production, and glucose-stimulated insulin secretion. These advantages may be provided by islet-derived CD146-positive stellate cells. The efficacy of xenotransplantation using islets cultured for a long time at 37°C was similar to that of overnight-cultured islets. In conclusion, 37°C might be a suitable temperature for the long-term culture of porcine islets, but further modifications will be required for successful xenotransplantation in a clinical setting.

In Vitro Evaluation of Intestinal Transport and High-Density Fermentation of Lactobacillus acidophilus.

Lactobacillus acidophilus strains have limiting factors such as low cell density and complex nutritional requirements in industrial production, which greatly restricts their industrial application. In this study, fermentation conditions for L. acidophilus were optimized and transcriptomic analysis used to understand growth mechanisms under high-density fermentation conditions. We found that L. acidophilus IMAU81186 has strong tolerance to gastrointestinal juice. In addition, its optimal culture conditions were 3% inoculum (v/v); culture temperature 37 °C; initial pH 6.5; and medium composition of 30.18 g/L glucose, 37.35 g/L soybean peptone, 18.68 g/L fish peptone, 2.46 g/L sodium citrate, 6.125 g/L sodium acetate, 2.46 g/L K2HPO4, 0.4 g/L MgSO4·7H2O,0.04 g/L MnSO4·5H2O, 0.01 g/L serine, and 0.3 g/L uracil. After optimization, viable counts of IMAU81186 increased by 7.03 times. Differentially expressed genes in IMAU81186 were analyzed at different growth stages using transcriptomics. We found that a single carbon source had limitations in improving the biomass of the strain, and terP and bfrA were significantly down-regulated in the logarithmic growth period, which may be due to the lack of extracellular sucrose. After optimizing the carbon source, we found that adding 12 g/L sucrose to the culture medium significantly increased cell density.

Increase CO2 recycling of Escherichia coli containing CBB genes by enhancing solubility of multiple expressed proteins from an operon through temperature reduction.

One of the current promising solutions to address problems of CO2 emissions is the development of biological carbon fixation strategies. This strategy leads to higher chemical biosynthetic productivity through improved biological CO2 fixation. In a previous study, we introduced the Calvin-Benson Bassham (CBB) genes of the photosynthetic bacterium Cereibacter sphaeroides into Escherichia coli to develop a strain capable of endogenous CO2 recycling by heterologous expression of the CBB genes. However, the heterologous expression of recombinant proteins in E. coli is often hampered by inclusion bodies (IB), which are protein aggregates. Various factors contribute to IB formation, including host cell metabolism, protein synthesis, transformation machinery, and target protein properties. In this study, we investigated the influence of environmental conditions, particularly culture temperature, on IB formation and CO2 recycling in the CBB strain. As a result, by reducing the culture temperature from 37°C to 30°C, a significant suppression of IB formation was achieved, resulting in a remarkable decrease in CO2 release by approximately 5.76 times. In addition, interestingly, an enhancement in the accumulation of pyruvate by approximately 2.3 times was observed at the same time. These results demonstrate the simultaneous improvements in CO2 recycling and the synthesis of organic acids achieved through temperature control. Based on the findings of this study, we believe that temperature control would be a promising approach to increasing chemical biosynthetic productivity as well as biological CO2 fixation activity in integrated autotrophic biorefinery strategies. IMPORTANCE In a previous study, we successfully engineered Escherichia coli capable of endogenous CO2 recycling through the heterologous expression of the Calvin-Benson Bassham genes. Establishing an efficient gene expression environment for recombinant strains is crucial, on par with the importance of metabolic engineering design. Therefore, the primary objective of this study was to further mitigate greenhouse gas emissions by investigating the effects of culture temperature on the formation of inclusion bodies (IB) and CO2 fixation activity in the engineered bacterial strain. The findings demonstrate that lowering the culture temperature effectively suppresses IB formation, enhances CO2 recycling, and concurrently increases the accumulation of organic acids. This temperature control approach, without adding or modifying compounds, is both convenient and efficient for enhancing CO2 recycling. As such, additional optimization of various environmental parameters holds promise for further enhancing the performance of recombinant strains efficiently.

Circadian gene transcription plays a role in cellular metabolism in hibernating brown bears, Ursus arctos.

Hibernation is a highly seasonal physiological adaptation that allows brown bears (Ursus arctos) to survive extended periods of low food availability. Similarly, daily or circadian rhythms conserve energy by coordinating body processes to optimally match the environmental light/dark cycle. Brown bears express circadian rhythms in vivo and their cells do in vitro throughout the year, suggesting that these rhythms may play important roles during periods of negative energy balance. Here, we use time-series analysis of RNA sequencing data and timed measurements of ATP production in adipose-derived fibroblasts from active and hibernation seasons under two temperature conditions to confirm that rhythmicity was present. Culture temperature matching that of hibernation body temperature (34°C) resulted in a delay of daily peak ATP production in comparison with active season body temperatures (37°C). The timing of peaks of mitochondrial gene transcription was altered as were the amplitudes of transcripts coding for enzymes of the electron transport chain. Additionally, we observed changes in mean expression and timing of key metabolic genes such as SIRT1 and AMPK which are linked to the circadian system and energy balance. The amplitudes of several circadian gene transcripts were also reduced. These results reveal a link between energy conservation and a functioning circadian system in hibernation.

Calibration of the carbonate clumped isotope thermometer of land snail shells

The application of carbonate clumped isotopes (Δ47) as a temperature proxy has been widely investigated in many types of carbonate materials, including biogenic carbonates such as foraminifera and corals. However, their application to land snails is relatively rare and it remains controversial whether shell carbonate Δ47 is impacted by biologically-driven fractionation. These uncertainties may stem from the lack of precise temperature-controlled calibration samples in previous natural-environment-based studies. In this study we cultured two species of land snail with different environmental tolerances: the thermo-humidiphilous Achatina fulica (A. fulica), and the cold-aridiphilous Cathaica fasciola (C. fasciola), under strictly controlled temperature conditions, covering the growth temperature range of 15–33 °C. The results show that the shell Δ47 values for both A. fulica and C. fasciola had statistically significant negative correlations with the culturing temperature within their respective optimum temperature ranges (A. fulica: 25–33 °C, C. fasciola: 15–25 °C). However, shell Δ47 values for A. fulica at lower temperatures (20 °C in this study) and those for C. fasciola showed a negative deviation from the expected equilibrium values. We attribute the lower-than-equilibrium values to the existence of a vital effect in individual snail species, and more specifically, to the CO2 degassing associated with dehydration/dehydroxylation during carbonate precipitation in the snail's calcifying fluid.

Dextranase Production Using Marine Microbacterium sp. XD05 and Its Application.

Dextranase, also known as glucanase, is a hydrolase enzyme that cleaves α-1,6 glycosidic bonds. In this study, a dextranase-producing strain was isolated from water samples of the Qingdao Sea and identified as Microbacterium sp. This strain was further evaluated for growth conditions, enzyme-producing conditions, enzymatic properties, and hydrolysates. Yeast extract and sodium chloride were found to be the most suitable carbon and nitrogen sources for strain growth, while sucrose and ammonium sodium were found to be suitable carbon and nitrogen sources for fermentation. The optimal pH was 7.5, with a culture temperature of 40 °C and a culture time of 48 h. Dextranase produced by strain XD05 showed good thermal stability at 40 °C by retaining more than 70% relative enzyme activity. The pH stability of the enzyme was better under a weak alkaline condition (pH 6.0-8.0). The addition of NH4+ increased dextranase activity, while Co2+ and Mn2+ had slight inhibitory effects on dextranase activity. In addition, high-performance liquid chromatography showed that dextran is mainly hydrolyzed to maltoheptanose, maltohexanose, maltopentose, and maltootriose. Moreover, it can form corn porous starch. Dextranase can be used in various fields, such as food, medicine, chemical industry, cosmetics, and agriculture.

Postprandial metabolism of Australian hybrid abalone (Haliotis laevigata × H. rubra) in relation to temperature and dietary protein manipulation

Understanding the physiological responses of abalone to culture conditions and nutritional inputs is paramount to maintain optimal performance under densely stocked farming conditions. High water temperature (>24 °C) is widely accepted as a key contributor to elevated summer mortality. High crude protein levels in modern abalone diets may interact with elevated water temperature to produce detrimental effects on abalone physiological processes but have received scant attention. This study measured the energy used in the digestion, absorption, and assimilation (i.e., specific dynamic action (SDA)) of hybrid abalone (Haliotis laevigata x H. rubra) after four months of acclimation to three protein levels (32, 38 and 44%) at three culture temperatures (12, 17 and 22 °C). Hybrid abalone exhibited significant stepwise increases in meal size as temperature increased from 12 through to 22 °C, contributing to significantly different SDA between the temperature treatments. Resting (standard) aerobic metabolic rate and peak metabolism (peak ṀO2) during SDA both increased with temperature, while SDA duration was lowest at 17 °C. No effect of dietary protein was observed within the temperature treatments for the SDA metrics. Abalone held at 12 °C exhibited significantly higher rates of gonad maturation, particularly among females. When 22 °C-acclimated abalone were exposed to a simulated summer heatwave up to 26 °C, they exhibited similar mortality and metabolic responses across protein treatment groups and there was a trend of steadily decreasing ṀO2 with sustained 26 °C water temperature, probably due to fasting. These results help to inform best farming practices to ensure optimal diet, growth, metabolic performance, and survival of hybrid abalone in the dynamic farm environment.

Optimisation of culture conditions for gesho (Rhamnus prinoides.L) callus differentiation using Artificial Neural Network-Genetic Algorithm (ANN-GA) Techniques

Gesho (Rhamnus prinoides) is a medicinal plant with antioxidant and anti-inflammatory activities commonly used in the ethnomedicinal systems of Africa. Using a three-layer neural network, four culture conditions viz., concentration of agar, duration of light exposure, temperature of culture, and relative humidity were used to calculate the callus differentiation rate of gesho. With the ability to quickly identify optimal solutions using high-speed computers, synthetic neural networks have emerged as a rapid, reliable, and accurate fitting technique. They also have the self-directed learning capability that is essential for accurate prediction. The network's final architecture for four selected variables and its performance has been confirmed with high correlation coefficient (R2, 0.9984) between the predicted and actual outputs and the root-mean-square error of 0.0249, were developed after ten-fold cross validation as the training function. In vitro research had been conducted using the genetic algorithm’s suggestions for the optimal culture conditions. The outcomes demonstrated that the actual gesho differentiation rate was 93.87%, which was just 1.86% lesser than the genetic algorithm's predicted value. The projected induced differentiation rate was 87.62%, the actual value was 84.79%, and the predicted value was 2.83% higher than Response Surface Methods optimisation. The environment for the growth of plant tissue can be accurately and efficiently optimised using a genetic algorithm and an artificial neural network. Further biological investigations will presumably utilise this technology.

A Simple and Effective Model for Predicting the Thermal Energy Requirements of Greenhouses in Europe

In this paper, a simple model is proposed for predicting the thermal energy requirements of greenhouses in Europe. The model estimates the annual heating requirements and the maximum required heating power, along with the corresponding heating and zero-energy operating periods. It is based on the greenhouse technical data (the overall heat loss coefficient, cover transmission, sensible absorbance), the cultivation conditions (temperature range), and the meteorological data (solar radiation and ambient temperature) according to the site characteristics (longitude and latitude). The model results can be obtained using a hand calculator. The model results are compared with those of a detailed model simulating a greenhouse’s thermal performance and they agreed with real data from the literature. Moreover, a model sensitivity analysis revealed the effect of various factors on the greenhouse’s energy requirements. The results proved that the most significant factor affecting heating requirements, the maximum heating power, and heating periods is the latitude of the greenhouse site, whereas zero-energy periods are primarily influenced by the plant cultivation temperature range and then by the latitude. According to our findings, in lower latitudes (40 to 50 degrees), heating requirements range from 250 to 430 kWh/m2/y, whereas, in higher latitudes (50 to 60 degrees), heating needs range from 430 to 650 kWh/m2/y.

The pathway of Edwardsiella piscicida infecting Lateolabrax maculatus via the immersion bath.

Edwardsiella piscicida, an infectious bacterium, causes great economic losses to the aquaculture industry. Immersion bath which is the closest way to how the fish infect bacterial pathogens in the natural environment is an effective route of artificial infection. In this study, the dynamic process of E. piscicida infection, in the spotted sea bass (Lateolabrax maculatus) was evaluated via the immersion bath. The results showed that soaking the spotted sea bass with 3 × 106 CFU mL-1 E. piscicida for 30 min could artificially induce edwardsiellosis. The higher culture temperature (28.5 ± 0.5°C) or the longer bath time (30 min) would lead to higher mortality of fish. E.piscicida first invaded the gill, then entered the blood circulation to infect the spleen and kidney, where it is colonized, and gradually multiplied in the liver and brain. Meanwhile, the fluorescence in situ hybridization showed that the localization of E. piscicida in the gill and foregut after the immersion challenge proceeded from the exterior to the interior. The invasion of pathogens triggers the immune response of fish and causes tissue damage to the host. The quantitative real-time PCR results displayed an increase in the relative expression level of immune genes (NK-lysin, LZM, IgM and IgD). Otherwise, the most notable histopathological changes of the infected spotted sea bass were multifocal necrosis. Findings in this study broaden our understanding of the infection conditions of E. piscicida and its pathogenicity to the spotted sea bass.