Optimal Culture Conditions Research Articles

Isolation, Identification, and Carbonate Mineralization Characteristics of a Newly Carbonic Anhydrase-Producing Strain.

Carbonic anhydrase-producing microorganisms can rely on their metabolism for carbon sequestration and carbonate precipitation, which is a relatively effective mode among the known microbially induced carbonate precipitation (MICP) methods. A newly carbonic anhydrase-producing strain was isolated from soil samples. 16S rDNA gene sequencing showed this strain had 99.18% sequence identity to Chryseobacterium gambrini. Various culture parameters (temperature, pH, rotational speed, inoculum size, and metal ions) were optimized for optimal microbial growth and CA activities. Optimal culture conditions were as follows: temperature of 30°C, pH 6-7, rotational speed 150rpm, and inoculum size 1%. It was observed that Co2+ and Mn2+ can improve CA activity with optimal concentrations of 0.02mM and 0.01mM, respectively. Furthermore, the introduction of CO2 for 15min daily leads to a 36% increase in the final production of biotic CaCO3, reaching 2.884g/L. Characterization of the mineralization precipitates was conducted to reveal the mechanism of the carbonic anhydrase-producing bacterium. Lastly, an analysis of the crystalline species and content of the biogenic CaCO3 was performed to lay the groundwork for future crystalline adjustments and to offer technical support for the application of the calcium method.

Production and Optimization of Extracellular α-amylase From Halophilic Bacteria Cytobacillus oceanisediminis Isolated From Wadi-El-Natrun, Egypt

The current study aimed to maximize α-amylase enzyme production by halophilic bacteria Cytobacillus oceanisediminis isolated from soil of Wadi-El-Natrun, Egypt. The results showed that of 21 halophilic bacterial isolates recovered from saline soil samples, eight isolates were high amylase producers. Interestingly, the bacterial isolate AHB6 exhibited the highest extracellular amylase production and was selected for further studies. This isolate was molecularly identified based on the 16srRNA gene sequencing as Cytobacillus oceanisediminis and has the accession number OM759999. The optimum temperature for α-amylase enzyme was 45 °C and exhibited thermostability at 45 °C for 1 h and retained more than 80% of its original activity. The α-amylase enzyme was active in almost all tested pHs and reached its highest activity at pH 9. Also, the optimum culture conditions for α-amylase enzyme production were wheat bran as carbon source, 2 g/l K2HPO4 as potassium source, 6 g/l MgSO4 as magnesium source and 0.5 g/l KNO3 as nitrogen source. These findings suggest the applicability of bacterial isolate Cytobacillus oceanisediminis as a potent producer of α-amylase for industrial purposes. Keywords: Amylase; Optimization; Cytobacillus oceanisediminis; Starch hydrolysis; Thermo-halophilic bacteria.

Systematic optimization and evaluation of culture conditions for the construction of circulating tumor cell clusters using breast cancer cell lines

BackgroundCirculating tumor cell (CTC) clusters play a critical role in carcinoma metastasis. However, the rarity of CTC clusters and the limitations of capture techniques have retarded the research progress. In vitro CTC clusters model can help to further understand the biological properties of CTC clusters and their clinical significance. Therefore, it is necessary to establish reliable in vitro methodological models to form CTC clusters whose biological characteristics are very similar to clinical CTC clusters.MethodsThe assays of immunofluorescence, transmission electron microscopy, EdU incorporation, cell adhension and microfluidic chips were used. The experimental metastasis model in mice was used.ResultsWe systematically optimized the culture methods to form in vitro CTC clusters model, and more importantly, evaluated it with reference to the biological capabilities of reported clinical CTC clusters. In vitro CTC clusters exhibited a high degree of similarity to the reported pathological characteristics of CTC clusters isolated from patients at different stages of tumor metastasis, including the appearance morphology, size, adhesive and tight junctions-associated proteins, and other indicators of CTC clusters. Furthermore, in vivo experiments also demonstrated that the CTC clusters had an enhanced ability to grow and metastasize compared to single CTC.ConclusionsThe study provides a reliable model to help to obtain comparatively stable and qualified CTC clusters in vitro, propelling the studies on tumor metastasis.

Evaluation of chemical attributes of soils: definition of management zones in silvipastoral system

Soil chemical analysis is a fundamental to achieve optimal cultivation conditions in a productive silvopastoral system, essential for the planning and management of this activity. The objective of this research is to map the spatial distribution of nutrients in the soil in a 5-year-old silvopastoral system composed of brachiaria-brizantha and jacarandá-da-bahia, and to analyze the effect of tree density and canopy on nutrient levels. The study was conducted at the experimental area of the Capixaba Institute for Research, Technical Assistance, and Rural Extension (INCAPER) in the district of Pacotuba, ES. The delimitation of the work area was done using geographic information systems, to create a regular grid of points to enable systematic soil sampling. A total of 75 disturbed soil samples were collected at depths of 0-0.10 m to obtain quantitative soil nutrient data. High-quality images obtained using a remotely piloted aircraft (RPA) allowed for the overlay of soil nutrient data, pasture data, forest inventory data, and sample plot data to assist in comparative analyses. This enabled identification of the areas with lower canopy development, particularly in the northeast portion. By cross-referencing the spatialized soil nutrient data with the data obtained through RPA, it was possible to correlate them with the lower or higher development of the system. The use of RPA, together with a georeferenced database, proved to be an innovative and powerful tool to support decision-making, allowing for the monitoring of system evolution and serving as a basis for evaluating the relationship between chemical elements and tree canopies. Keywords: geostatistics, precision agriculture, soil nutrients.

Screening and Optimization of IAA Production by PGPR isolated from Rhizosphere of a Pterocarpus marsupium Roxb. and their Effect on Plant Growth

Indole Acetic Acid (IAA) production is important attribute of PGPR that promote plant growth and development. The rhizosphere is hotspot in the soil that harbors PGPR. The present study was aimed with isolation and screening of IAA producing bacteria from the rhizosphere of Pterocarpus marsupium Roxb. Optimum culture conditions (pH, temperature, incubation period and L-tryptophan concentration for IAA production were studied for selected isolates and their effect on wheat growth and root development was evaluated. Among twenty four IAA producing isolates five isolates (Et1, Rp1, Rp5, Rp6, and Rp9) produced maximum IAA in range of 50-70 μg/mL and was used in optimization studies. Maximum IAA was produced in 96 hours of incubation, at pH 7 and with 0.1mg/mL of L-tryptophan by all five isolates. 30oC is the most suitable temperature for Et1, Rp1, Rp5, Rp9; whereas Rp6 produced nearly same amount of IAA at wide range of temperature 30-35oC (77-84.12 μg/mL) and at pH 7-8 ( 73-74μg/mL). Out of the five isolates, Rp6 exhibits the highest potential, having a maximum IAA of 84.12 μg/mL at 35°C and pH 7. Although tryptophan influences IAA synthesis but at higher concentration of tryptophan inhibits IAA synthesis. To validate the production of IAA, crude extracts were analyzed using thin layer chromatography (TLC). A spot of standard IAA with the same Rf value (0.91) was found to match a specific spot from the crude IAA.

Green alternatives to petroleum-based plastics: production of bioplastic from Pseudomonas neustonica strain NGB15 using waste carbon source.

Polyhydroxyalkanoates have attracted great interest as a suitable alternative to petrochemical based plastics due to their outstanding properties such as biodegradability and biocompatibility. However, the biggest problem in the production of microbial polyhydroxyalkanoates is low cost-effectiveness. In this study, polyhydroxyalkanoate production was carried out using waste substrates with local isolates. Culture conditions were optimized to increase the polyhydroxyalkanoate production potential. The produced polyhydroxyalkanoate was characterized by FTIR analyses, and its metabolic pathway was determined by real-time PCR. According to the results, the best polyhydroxyalkanoate producer bacteria was characterized as Pseudomonas neustonica NGB15. The optimal culture conditions were detected as 30g/L banana peel powder, 25°C temperature, pH 8, and 4-day incubation time. Under the optimized conditions, 3.34g/L PHA production was achieved. As a result of FTIR analyses, major peaks were obtained at 1723, 1277, 1261, 1097, 1054, and 993cm-1. These peaks represent that the type of produced polyhydroxyalkanoate was poly-β-hydroxybutyrate. According to gene expression profile of NGB15, it was determined that Pseudomonas neustonica NGB15 produces PHA using the de novo fatty acid synthesis metabolic pathway. In conclusion, poly-β-hydroxybutyrate production by Pseudomonas neustonica NGB15 using a low-cost fermentation medium has been shown to be biotechnologically promising.

Recombinant Production of Hydrophobin DewA in Pichia pastoris and Determination of Its Functions

Background Hydrophobins have great potential in many biotechnological applications due to changing surface characteristics. In recent years, although there has been a significant increase in the biotechnological applications of hydrophobins, industrial production has still not been achieved due to yield problems. Therefore, more studies are needed on the recombinant production of hydrophobins. In this work, the recombinant production of class I hydrophobin DewA from Aspergillus nidulans, which is determined to have high contact angle in the literature, was aimed. As a result, DewA protein was recombinantly produced using P. pastoris X-33 strain under AOX1 promoter by transferring into pPICZα-A vector. Results The optimal culture condition for DewA expression was obtained at 1% methanol concentration as 77 mg/L in 96 hour. Recombinant DewA has been proven to change the surface characteristics on the teflon and glass surfaces. Then, the surface stability of the protein was evaluated by applying hot SDS and UV to these surfaces. The surface-coated DewA was resistant to hot SDS application on both glass and teflon surfaces; in the UV application, it was understood that while the protein was degraded by UV exposure on glass surfaces, it preserved its structure on teflon surfaces. Conclusions In the study, the DewA protein of A.nidulans was cloned into the pPICZα-A vector and recombinantly produced in the P.pastoris X-33 strain for the first time.

Comprehensive assessment of goat adiposetissue-derived mesenchymal stem cells cultured in different media.

Mesenchymal stem cells (MSCs) have demonstrated potential in treating livestock diseases that are unresponsive to conventional therapies. MSCs derived from goats, a valuable model for studying orthopaedic disorders in humans, offer insights into bone formation and regeneration. Adipose tissue-derived MSCs (ADSCs) are easily accessible and have a high capacity for expansion. Although the choice of culture media significantly influences the biological properties of MSCs, the optimal media for goat ADSCs (gADSCs) remains unclear. This study aimed to assess the effects of four commonly used culture media on gADSCs' culture characteristics, stem cell-specific immunophenotype, and differentiation. Results showed that MEM, DMEM/F12, and DMEM-LG were superior in maintaining cell morphology and culture parameters of gADSCs, such as cell adherence, metabolic activity, colony-forming potential, and population doubling. Conversely, DMEM-HG exhibited poor performance across all evaluated parameters. The gADSCs cultured in DMEM/F12 showed enhanced early proliferation and lower apoptosis. The cell surface marker distribution exhibited superior characteristics in gADSCs cultured in MEM and DMEM/F12. In contrast, the distribution was inferior in gADSCs cultured in DMEM-LG. DMEM/F12 and DMEM-LG culture media demonstrated a significantly higher potential for chondrogenic differentiation and DMEM-LG for osteogenic differentiation. In conclusion, DMEM/F12 is a suitable culture medium for propagating gADSCs as it effectively maintains cell morphology, growth parameters, proliferation and lower apoptosis while exhibiting desirable expression patterns of MSC-specific markers. These findings contribute to optimising culture conditions for gADSCs, enhancing their potential applications in disease treatment and regenerative medicine.

Identification, Nutrient Composition, and Evaluation of a Wild Pleurotus citrinopileatus Strain (X21156) from Tibet for Antioxidant and Cytotoxic Activities

A fungal strain (X21156) collected in Tibet was used as the material, identified based on its morphological characteristics and internal transcribed spacer (ITS) sequence; its optimal culture conditions were analyzed by single-factor experiments; artificial domestication and cultivation were carried out; its nutrient composition was determined; and the bioactivities of its polysaccharides were detected using chemical antioxidant assays and MTT assays. The results showed that the strain was Pleurotus citrinopileatus Sing. Its optimal culture conditions were a pH of 7, a temperature of 25 °C, glucose (20 g·L−1) as the carbon source, and yeast powder (20 g·L−1) as the nitrogen source. The fresh weight of a single domesticated fruiting body was 41.16 g. The strain had high protein (28.5%), high fiber (34%), and low fat (1.4%) contents, with high proportions of fresh and sweet amino acids. Polysaccharides had good scavenging ability on ABTS+, DPPH, and OH free radicals (EC50 0.06 mg/mL, 1.21 mg/mL, and 3.62 mg/mL, respectively), and the cytotoxicity of polysaccharides to hepatocellular carcinoma cells (HepG2) (IC50: 1.69 mg/mL) was higher than that of triple-negative breast cancer cells (MDA-MB-468) (IC50: 1.76 mg/mL). In conclusion, the study provides a reference on the optimal culture conditions, domestication and cultivation, and dietary and medicinal values of wild P. citrinopileatus Sing.

Synergistic analysis of lignin degrading bacterial consortium and its application in rice straw fiber film

Fiber film have received widespread attention due to its green friendliness. We can use microorganisms to degrade lignin in straw to obtain cellulose and make fiber films. Herein, a group of high-temperature (50 °C) lignin degrading bacterial consortium (LDH) was enriched and culture conditions for lignin degradation were optimized. Combined with high-throughput sequencing technology, the synergistic effect of LDH-composited bacteria was analyzed. Then LDH was used to treat rice straw for the bio-pulping experiment. The results showed that the lignin of rice straw was degraded 32.4 % by LDH at 50 °C for 10 d, and after the optimization of culture conditions, lignin degradation rate increased by 9.05 % (P < 0.001). The bacteria that compose in LDH can synergistically degrade lignin. Paenibacillus can encode all lignin-degrading enzymes present in the LDH. Preliminary tests of LDH in the pulping industry have been completed. This study is the first to use high temperature lignin degrading bacteria to fabricate fiber film.

Impact of irradiance and inorganic carbon availability on heterologous sucrose production in Synechococcus elongatus PCC 7942.

Cyanobacteria have been proposed as a potential alternative carbohydrate feedstock and multiple species have been successfully engineered to secrete fermentable sugars. To date, the most productive cyanobacterial strains are those designed to secrete sucrose, yet there exist considerable differences in reported productivities across different model species and laboratories. In this study, we investigate how cultivation conditions (specifically, irradiance, CO2, and cultivator type) affect the productivity of sucrose-secreting Synechococcus elongatus PCC 7942. We find that S. elongatus produces the highest sucrose yield in irradiances far greater than what is often experimentally utilized, and that high light intensities are tolerated by S. elongatus, especially under higher density cultivation where turbidity may attenuate the effective light experienced in the culture. By increasing light and inorganic carbon availability, S. elongatus cscB/sps produced a total of 3.8 g L-1 of sucrose and the highest productivity within that period being 47.8 mg L-1 h-1. This study provides quantitative description of the impact of culture conditions on cyanobacteria-derived sucrose that may assist to standardize cross-laboratory comparisons and demonstrates a significant capacity to improve productivity via optimizing cultivation conditions.

Potential of hospital wastewater treatment using locally isolated Chlorella sp. LH2 from cocoon wastewater

Chlorella sp. is able to grow and transform inorganic and organic contaminants in wastewater to create biomass. In the present study, Chlorella sp. LH2 isolated from cocoon wastewater was able to thrive in hospital wastewater, then remove nutrients and eliminate E. coli ATCC 8739. The results indicated that optimal cultivation conditions of Chlorella sp. LH2 in hospital wastewater were pH of 8, light:dark cycle of 16:8 at 30oC. The inhibitory effect of chlorination on algae growth was accompanied with the chlorine concentration. BOD5:COD ratio of 0.77 indicated biodegradability of hospital wastewater. The untreated and treated wastewatee samples were collected to investigated the nutrient removal efficiency after 10 days. Untreated and treated results were192 ± 8.62 mg/l 23.91 ± 2.19 mg/l for BOD5; 245 ± 9.15 mg/l and 47.31 ± 5.71 mg/l for COD. The treated value met the required standards for hospital wastewater treatment. The removal efficiency total nitrogen and total phosphorus were 68.64% and 64.44% after 10 days, respectively. Elimination of E. coli ATCC 8739 after 7 days by Chlorella sp. LH2 was 88.92%. The results of this study suggest the nutrients and pathogens removal potential of Chlorella sp. LH2 in hospital wastewater for further practical applications.

Genome analysis of a newly isolated Bacillus velezensis-YW01 for biodegrading acetaldehyde.

Acetaldehyde (AL), a primary carcinogen, not only pollutes the environment, but also endangers human health after drinking alcohol. Here a promising bacterial strain was successfully isolated from a white wine cellar pool in the province of Shandong, China, and identified as Bacillus velezensis-YW01 with 16S rDNA sequence. Using AL as sole carbon source, initial AL of 1g/L could be completely biodegraded by YW01 within 84h and the cell-free extracts of YW01 has also been detected to biodegrade the AL, which indicate that YW01 is a high-potential strain for the biodegradation of AL. The optimal culture conditions and the biodegradation of AL of YW01 are at pH 7.0 and 38°C, respectively. To further analyze the biodegradation mechanism of AL, the whole genome of YW01 was sequenced. Genes ORF1040, ORF1814 and ORF0127 were revealed in KEGG, which encode for acetaldehyde dehydrogenase. Furthermore, ORF0881 and ORF052 encode for ethanol dehydrogenase. This work provides valuable information for exploring metabolic pathway of converting ethanol to AL and subsequently converting AL to carboxylic acid compounds, which opened up potential pathways for the development of microbial catalyst against AL.

Antioxidant and anti-stress properties of postbiotics produced by Lysinibacillus macroides G117

In this study, Lysinibacillus macroides G117 was isolated from vinegar residue, and the probiotic activity of its postbiotics was determined in Artemia salina and Scophthalmus maximus. First, the functional genome of L. macroides G117 was analysed. The total length of the genome was 4,728,501 bp, composed of one ring chromosome and two ring plasmids. Two gene clusters, β-lactone and type III polyketide synthases (T3PKS), well-known for anti-toxic, anti-inflammatory, antioxidant, anticancer activities, were predicted through the antiSMASH database. In addition, probiotic genes for antioxidants, stress resistance, and anaerobic respiration were identified using the KEGG database. These results suggest that L. macroides G117 could be used as a probiotic in aquaculture. To further assess the probiotic properties of L. macroides G117, postbiotics were cultured and collected. Based on the survival rate of pesticide-stimulated A. salina, the optimum culture condition of L. macroides G117 was analysed. The optimal culture conditions were 1 % sucrose, 1.5 % yeast extract, 0.1 % MgSO4·7H2O, 30 °C, pH 7.8, bottled fluid volume 100 mL, shaking speed 180 rpm, inoculation quantity 0.5 %, and fermentation for 48 h, with a 15 % increase in survival rate of A. salina with the addition of optimised postbiotics. Untargeted metabolomic analysis revealed significant differences between the non-optimised and optimised postbiotics, mainly in amino acid and carbohydrate metabolites. Notably, postbiotics significantly increased the antioxidant enzymes in A. salina (catalase (CAT) and total superoxide dismutase (T-SOD)) and S. maximus (T-SOD) and decreased malondialdehyde (MDA) content. In addition, the activities of anaerobic respiratory-related enzymes (lactate dehydrogenase (LDH) and pyruvate kinase (PK)) were significantly increased, whereas those of aerobic respiratory-related enzymes (malate dehydrogenase (MDH)) were significantly decreased in S. maximus. These results suggest that L. macroides G117 postbiotics could improve the antioxidant and anti-stress abilities of the two types of organisms.

Amelioration of L-methionine production by Alcaligenes faecalis ATCXT3624: Empirical optimization of culture conditions

Background: L-methionine production at the industrial scale suffers from various drawbacks, including the production and separation of the L-enantiomer of the amino acid methionine by chemical production methods. This strain exhibiting resilience to feedback inhibition by L-methionine provided a promising avenue for enhanced production. The present study has tried to uplift the production rate of the amino acid by a developed L-methionine-resistant strain Alcaligenes faecalis ATCXT 3624 through surpassing feedback inhibition. Methods: Empirical optimization of all fermentation conditions was studied by observing the effects of initial pH, temperature, inoculum age, medium volume, cell density, and different micronutrients, which enhanced L-methionine production. In addition, efforts were made to find suitable nitrogen and carbon sources. Results: Eventually, 23.8±0.22 mg/mL of L-methionine was obtained under optimized fermentation conditions, which is significantly (p&lt;0.05) higher than that produced before optimized fermentation conditions. Conclusion: Eventually, it can be assumed that the strain effectively produced the amino acid at an enhanced rate under optimized conditions.

Coral mariculture using abandoned abalone farming ponds in northeastern Taiwan

Stony corals are ecologically and economically important marine organisms. Coral aquaculture is essential to provide live coral materials for the marine ornamental trade, reef restoration programs, and environmental education. This study sought to establish coral farms in Taiwan. To culture corals, we adopted an abandoned abalone farming pond built along the northeastern coast of Taiwan, and investigated whether it is possible to propagate corals in this artificial environment. Stainless steel underwater tables were anchored to the pond floor, and fragments of Acropora tumida, Pocillopora damicornis, Stylophora pistillata, Favites pentagona, and Porites lobata were cultured on the tables for one year. Environmental factors in the pond were also monitored. The mean surface temperature was 23.07 °C, ranging from >30 °C in summer to 18 °C in winter. The mean salinity and pH were about 35 ppt and 8.14, respectively. Survivorship of these five species after one year ranged from 100% to 67%, with a 1.37–1.51-fold increase in weight and a 3.22–3.38-fold increase in area, demonstrating that although environmental factors fluctuate depending on the season and weather, long-term and stable asexual propagation of corals is feasible in the pond. For further optimization of culture conditions, the effects of initial fragment size for culture on subsequent survival and growth were investigated. Fragments of 3 sizes (1.5, 3.0, and 4.5 cm) were prepared from parental colonies of A. tumida, P. damicornis, and S. pistillata and cultured for 6 months by hanging them under the table. There were no significant differences in survivorship among these sizes. Differences were observed in biomass (weight) increase. In A. tumida, 3-cm fragments showed significantly more weight gain than other lengths, while 1.5-cm fragments showed a significant weight increase in P. damicornis. In S. pistillata, no significant difference in weight increase was observed among size groups, suggesting the necessity to optimize the initial fragment size for culture for each species before mass culture. Histological analysis showed that mature gametes were formed in some cultured corals, suggesting that sexual propagation may also be possible in the future. The established coral farm, which is culturing >4000 coral fragments now, will be able to support not only basic and applied coral research, but also sustainable reef development and marine ornamental trade.

An optimized protocol for routine development of cell culture from adult oyster, Crassostrea madrasensis.

Marine molluscan cell lines, required for virus screening and cultivation, form essential tools for developing health management strategies for these animals in the blue economy. Moreover, they are also crucial to develop cultivated seafood. As there is no valid marine molluscan cell line, primary cell cultures are relied upon for all investigations. A sound protocol for generating primary cell cultures from molluscs is entailed, but existing protocols often involve heavy antibiotic usage and depuration that invariably affect gene expression and cell health. This work presents an easy-to-adopt, time-saving protocol using non-depurated mollusc Crassostrea madrasensis, which requires only initial antibiotic treatment and minimal exposure or no use of antibiotics in the cell culture medium. The important experimental considerations for arriving at this protocol have been elucidated. Accordingly, sodium hypochlorite and neomycin sulfate were chosen for disinfecting tissues. The study is the first to use shrimp cell culture medium (SCCM) as a cell culture medium for molluscan cell culture. Despite being osmoconformers, the oysters exhibited stable intracellular osmotic conditions and pH, which, when provided in vitro, promoted effective cardiomyocyte formation. The cell viability could be enhanced using 10% fetal bovine serum (FBS), but healthy cell culture could also be obtained using SCCM without FBS. The optimized culture conditions allowed for regular beating cardiomyocyte clusters that could be retained for a month. Limited cell proliferation, as shown by the BrdU assay, demands further interventions, such as possibly producing induced pluripotent stem cells. The optimized protocol and culture conditions also align with some requirements for producing cultivated meat from marine molluscs.

Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota.

Sewer biofilms are likely to constitute hotspots for selecting and accumulating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to optimize culture conditions to obtain in vitro biofilms, mimicking the biofilm collected in sewers, to study the impact of fluoroquinolones (FQs) on sewer biofilm microbiota. Biofilms were grown on coupons in CDC Biofilm Reactors®, continuously fed with nutrients and inoculum (1/100 diluted wastewater). Different culture conditions were tested: (i) initial inoculum: diluted wastewater with or without sewer biofilm, (ii) coupon material: concrete vs. polycarbonate, and (iii) time of culture: 7 versus 14 days. This study found that the biomass was highest when in vitro biofilms were formed on concrete coupons. The biofilm taxonomic diversity was not affected by adding sewer biofilm to the initial inoculum nor by the coupon material. Pseudomonadales, Burkholderiales and Enterobacterales dominated in the sewer biofilm composition, whereas in vitro biofilms were mainly composed of Enterobacterales. The relative abundance of qnrA, B, D and S genes was higher in in vitro biofilms than sewer biofilm. The resistome of sewer biofilm showed the highest Shannon diversity index compared to wastewater and in vitro biofilms. A PCoA analysis showed differentiation of samples according to the nature of the sample, and a Procrustes analysis showed that the ARG changes observed were linked to changes in the microbial community. The following growing conditions were selected for in vitro biofilms: concrete coupons, initial inoculation with sewer biofilm, and a culture duration of 14 days. Then, biofilms were established under high and low concentrations of FQs to validate our in vitro biofilm model. Fluoroquinolone exposure had no significant impact on the abundance of qnr genes, but high concentration exposure increased the proportion of mutations in gyrA (codons S83L and D87N) and parC (codon S80I). In conclusion, this study allowed the determination of the culture conditions to develop an in vitro model of sewer biofilm; and was successfully used to investigate the impact of FQs on sewer microbiota. In the future, this setup could be used to clarify the role of sewer biofilms in disseminating resistance to FQs in the environment.

Optimization of Culture Conditions and Batch Process Control for the Augmented Production of Bacteriocin by Bacillus Species.

The emergence of antibiotic-resistant microorganisms poses a significant threat to human health worldwide. Recent advances have led to the discovery of molecules with potent antimicrobial activity from environmental sources. In this study, fifteen bacterial isolates were obtained from agricultural and polluted soil samples collected from different areas of the cities of Jizan and Jeddah. These isolates were screened for antagonistic activity against a set of human pathogenic bacterial strains. The results showed that two Bacillus strains, identified as Bacillus atrophaeus and Bacillus amyloliquefaciens based on 16S rDNA, synthesized bacteriocin with strong antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591, Pseudomonas aeruginosa ATCC 9027, Salmonella typhimum ATCC 14028, carbapenem-resistant E. coli, and MRSA 2. To optimize bacteriocin production, the effects of medium composition, incubation period, temperature, and pH were investigated. Nutrient broth and Mueller-Hinton broth were chosen as the optimal original media for bacteriocin production. The optimal incubation period, temperature, and pH were found to be 48 h at 37 °C and 7 pH in Bacillus atrophaeus and 72 h at 37 °C and 8 pH in Bacillus amyloliquefaciens. Batch cultures of Bacillus atrophaeus and Bacillus amyloliquefaciens were grown in a 10 L benchtop bioreactor, and pH control was found to significantly increase the production of bacteriocin by two-fold compared to uncontrolled conditions. The time course of growth, substrate consumption, pH, and enzyme production were investigated. This study demonstrates the potential of optimizing culture conditions and batch process control to enhance bacteriocin production by Bacillus spp.

Meadow clover is the most important forage crop in the western part of the Non-chernozem zone

Relevance. The task of modern feed production is to increase feed production, as well as improve their quality and energy saturation. A special place in solving this problem belongs to perennial legumes and grasses. At the same time, it is important to use precisely those crops and varieties that have the greatest biological potential.Methods. The objects of research were meadow clover (Pochinkovets variety and Topaz variety), timofeevka meadow (VIC 911 variety), ryegrass pasture (Karat variety and Express variety), for the realization of the biological potential of which solid mineral fertilizers (ammonium nitrate, double superphosphate, potassium chloride) are needed, as well as concentrated liquid complex fertilizers of the series “Ultramagus.”Results. Among the studied species of perennial grasses, meadow clover has the highest yield, so among the clover varieties, the Pochinkovets variety had the greatest potential. Varieties of meadow clover increased the collection of dry matter when applying solid phosphorus-potassium fertilizers and liquid complex fertilizer “Ultramag Combi”. The highest yield was obtained in variants with the combined use of root fertilization with solid fertilizers at the rate of P80K120 and foliar fertilization with liquid complex fertilizer “Ultramag Combi”. The yield of meadow clover, depending on the variety, ranged from 12.05 to 12.46 t/ha. Varieties of meadow clover Pochinkovets and Topaz in the conditions of the Smolensk region form a tall monodominant herbage capable of intensive regrowth during the season and yielding at least two full-fledged mowing under optimal cultivation conditions.