Optimal Culture Conditions Research Articles

Screening, Identification, and Optimization of Enzyme Production for Bacteria with High Efficiency of Bovine Blood Protein Degradation from Slaughterhouses

Blood waste originating from slaughterhouse activities in China is considered to be massive, and improper handling may cause epidemic diseases and environmental pollution. This research aimed to obtain a potential bacterial strain for blood protein degradation by isolating indigenous bacteria from slaughterhouses. Isolation was carried out by using dilution plate coating, while blood agar and casein plates were used to screen potential strains. Morphological, physiological, and biochemical characterizations, as well as 16S rRNA sequencing, were performed to identify the selected strain. The optimization of enzyme production was performed by using the Plackett–Burman test and response surface methodology. A strain coded NwMCC01910137 was isolated and screened to effectively degrade bovine blood protein and was identified as Bacillus subtilis. The optimum culture conditions for enzyme production included a fermentation temperature of 37.4 °C, an initial pH of 7.7, a soybean meal powder addition amount of 3.00% (w/v), a glucose level of 3.8% (w/v), a NaCl level of 0.3 g/L, a phosphate concentration of 2.5 g/L, an inoculum size of 2.5 g/L (4%), and a fermentation time of 48 h. Under the optimum condition, the strain showed enzyme activity of 166.83 U/mL. Hence, the isolated B. subtilis showed good activity in bovine blood protein degradation and has good application potential for slaughterhouse waste processing.

Tailored culture strategies to promote antimicrobial secondary metabolite production in Diaporthe caliensis: a metabolomic approach

BackgroundIn the search for new antimicrobial secondary metabolites of fungi, optimizing culture conditions remains a critical challenge, as standard laboratory approaches often result in low yields. While non-selective methods, such as modifying culture media, have been effective in expanding the chemical diversity of fungal metabolites, they have not yet established a direct link to key process parameters crucial for further optimization. This study investigates the capacity of Diaporthe caliensis as a biofactory for biologically active secondary metabolites, employing tailored culture media to explore the relationship between chemical diversity and critical process variables.ResultsThe metabolomic profiles, antibacterial activities, and production yields of the extracts were analyzed to progressively adjust the culture conditions. This study was conducted in five steps, evaluating carbon and nitrogen source concentration, nitrogen source type, salt supplementation, and pH adjustment. Altering the rice starch concentration affected biomass yield per unit of oxygen consumed, while modifications to the nitrogen source concentration influenced both the bioactivity and chemical space by Diaporthe caliensis. Despite changes at the metabolome level, the extracts consistently exhibited potent antibacterial activities, influenced by the nitrogen source, added salts and pH adjustments. For instance, when using corn steep liquor and rice starch, supplemented with micronutrients, different metabolites were produced depending on whether buffer or water was used, though both conditions showed similar antibacterial activities (IC50 ≈ 0.10 mg mL− 1 against Staphylococcus aureus and ≈ 0.14 mg mL− 1 against Escherichia coli). In the treatment where buffer was used to stabilize pH change, there was an increase in the production of phomol-like compounds which are associated with known antibiotic properties. In contrast, in the treatments using water, the drop in pH stimulated the production of previously unidentified metabolites with potential antimicrobial activity.ConclusionsThis study proposes a strategic methodology for the tailored formulation of culture media aiming to promote the biosynthesis of diverse secondary metabolites. This approach revealed the critical role of nutrient limitation and pH regulation in stimulating the production of polyketide-lactone derivatives, including the antibiotic phomol. Ultimately, the systematic, custom-designed culture conditions developed in this work offer a promising strategy for expanding the chemical diversity of Diaporthe caliensis, while providing valuable insights into the key parameters needed for optimizing this fungal biofactory.Graphical

Morphological and molecular identification of Schizophyllum commune causing storage bulb rot disease of Lanzhou edible lily in China and its biological characteristics

Lily bulb rot disease has harmed edible lily in recent years, resulting in yield losses in China. As a results, both morphological and molecular techniques must be used to confirm the etiology of storage bulb rot disease on lily bulbs. Lily bulbs with indications of rot symptoms during storage were gathered in Lanzhou, Gansu Province, China. A strain was isolated and its morphologically characterized as a Schizophyllum commune specie. Pathogenicity tests further confirmed that the strain caused apparent S. commune symptoms on lily bulbs which were consistent with those seen in the field. The pathogenicity of S. commune to lily bulb was 100%, and morphological identification showed that the mycelia of the pathogen were white and villous, with septate, branched, and acicular bodies and obvious lock-shaped joints. The mycelia had uneven thickness, ranging from 1.03 to 3.06 μm, and turned gray-white at the later growth stage. Moreover, the pathogen formed a mycelial column on potato dextrose agar (PDA) medium, which formed a coral-like fruiting body primordium, which split, forming pileus for spore production. The spores were colorless and cylindrical, with an oblique tip and a size of 4.6–7.2 μm × 2.2–2.7 μm. The isolate was deduced as based on phylogenetic analysis with 2 genes (ribosomal DNA-ITS and LSU) as well as morphological characteristics and cultural features, the isolate was identified S. commune. Soluble starch, yeast extract, temperature of 30°C, pH 7, relative humidity of 100%, and complete dark were shown to be the optimum culture conditions for surface mycelium growth. In conclusion, this is the first report of S. commune causing bulb rot of edible lilies in China. The study provides a basis for more effective control strategies for lily bulb rot disease.

Long-term in vitro expansion of a human fetal pancreas stem cell that generates all three pancreatic cell lineages

The mammalian pancreas consists of three epithelial compartments: the acini and ducts of the exocrine pancreas and the endocrine islets of Langerhans. Murine studies indicate that these three compartments derive from a transient, common pancreatic progenitor. Here, we report derivation of 18 human fetal pancreas organoid (hfPO) lines from gestational weeks 8-17 (8-17 GWs) fetal pancreas samples. Four of these lines, derived from 15 to 16 GWs samples, generate acinar-, ductal-, and endocrine-lineage cells while expanding exponentially for >2 years under optimized culture conditions. Single-cell RNA sequencing identifies rare LGR5+ cells in fetal pancreas and in hfPOs as the root of the developmental hierarchy. These LGR5+ cells share multiple markers with adult gastrointestinal tract stem cells. Organoids derived from single LGR5+ organoid-derived cells recapitulate this tripotency invitro. We describe a human fetal tripotent stem/progenitor cell capable of long-term expansion invitro and of generating all three pancreatic cell lineages.

GC-MS Analysis of Polysaccharides from an Intergeneric Hybrid of Pleurotus florida and Cordyceps militaris: A Comparative Study.

Edible and medicinal mushrooms are valuable sources of polysaccharides, known for their dual roles as immunostimulants and immunosuppressants. This study aimed to enhance polysaccharide content by fusing two mushroom species, P. florida and C. militaris, while exploring their antioxidant and antibacterial potential. These mushrooms have diverse health benefits, including lowering high cholesterol, providing anti-inflammatory effects, supporting diabetes management, aiding in cancer treatment, and enhancing the efficacy of COVID-19 vaccines. Successful hyphal fusion was achieved, and optimal culture conditions were determined using response surface methodology. The hybrids exhibited superior growth compared to the parental strains. Hyphal fusion improved several attributes, resulting in diverse hybrids with increased biomass and metabolite production. FTIR analysis confirmed the presence of exopolysaccharides, with concentrations measured at 28.4g/L (P1), 31.50g/L (CD), and 36.74g/L (F3). GC-MS analysis identified various bioactive metabolites, including a higher concentration of dimethyl palmitamine in the hybrid, a novel compound, butanenitrile, 2-(methoxymethoxy), which was not found in the parental strains. These compounds are likely responsible for the enhanced antimicrobial and antioxidant activities.

Comprehensive cultivation of the broiler gut microbiota guides bacterial isolation from chickens.

Chicken gut microbiota plays an important role in maintaining their physiological health. However, the cultivability of chicken gut microbiota is not well understood, limiting the exploration of certain key gut bacteria in regulating intestinal health and nutritional metabolism. This study aimed to examine the cultivability of chicken cecal microbiota and to provide guidance for future chicken gut microbiota cultivation. A total of 58 different culture conditions were applied to culture broiler cecal microbiota, and the culture-dependent (CD; pooled colonies form each plate) and culture-independent (CI; broiler cecal contents) samples were collected for 16S rRNA gene sequencing and microbial analysis. The CD methods detected higher microbial richness (3,636 vs 2,331 OTUs) than CI methods, and the recovery rates of bacterial OTUs and genera reached 43.6% and 68.9%, respectively. The genera of Bacteroides (19.9%), Alistipes (11.0%) and Barnesiella (10.7%) were highly abundant detected by CI methods, however, there occupied a small proportion (<1.0%) of total cultured microbiota in CD methods. We then developed reference figures and tables showing optimal cultivation conditions for different gut bacteria taxa. Moreover, 81 different lactic acid bacteria strains covering 5 genera were isolated, and 15 strains had less than 97.0% similarity to known bacteria in the national center for biotechnology information (NCBI) online database. Overall, this study provides preliminary guidance in culturing specific gut microbiota from chickens, which will contribute to future studies to characterize the biological functions of key microbes in chicken nutritional metabolism and health.

Optimization of culturing conditions for leafcutter ant associated, antimicrobial producing Pseudonocardia by adding ants´ cuticular hydrocarbons

Optimization of culturing conditions for leafcutter ant associated, antimicrobial producing Pseudonocardia by adding ants´ cuticular hydrocarbons

Light Intensity Effects on Productivity and Post-Harvest Quality in Perilla frutescens Cultivated in CEA

Leafy vegetables, mainly lettuces, are currently the main crop cultivated in controlled environment agriculture (CEA), including vertical farming and plant factories. There is a rising demand to expand this portfolio with a wider variety of underutilized edible plants containing various bioactive compounds and sensory properties seeking to enrich human diets. However, the optimal cultivation conditions for these underutilized plants significantly differ from those optimized for lettuce, basil, and other popular CEA crops. Therefore, this study aims to explore the impacts of light-emitting diode (LED) lighting intensity (photosynthetic photon flux density, PPFD) on green leaf Perilla frutescens cultivated in CEA. Plants were grown under four levels of LED lighting PPFDs from 150 to 300 µmol m−2s−1 for 4 weeks. Plant biomass productivity, soluble sugar contents, antioxidant properties (DPPH, ABTS free radical scavenging activities, FRAP antioxidant power), and total contents of phenolic compounds in leaves were evaluated at harvesting time. Further, harvested plant material was stored in the dark, at +6 °C, and the water content, water loss and transpiration rate, leaf sugar contents, and antioxidant properties were monitored 1, 3, and 5 days after harvesting. The summarized data suggest that higher cultivation lighting PPFD results in better harvest quality preservation during post-harvest storage.

Change in p53 nuclear localization in response to extracellular matrix stiffness

AbstractChondrocytes are commonly applied in regenerative medicine and tissue engineering. Thus, the discovery of optimal culture conditions to obtain cells with good properties and behavior for transplantation is important. In addition to biochemical cues, physical and biomechanical changes can affect the proliferation and protein expression of chondrocytes. Here we investigated the effect of extracellular matrix stiffness on mouse articular chondrocyte phenotype, growth, and subcellular p53 localization. Chondrocytes were seeded on collagen‐coated substrates varying in elasticity: 0.5 and 100 kPa. Immunocytochemical staining and immunoblotting showed that a softer substrate significantly increased p53 nuclear localization in chondrocytes. Furthermore, we identified microRNA‐532 (miR‐532) as a potential p53 target gene to influence cell function, indicating a new target for tissue engineering. These findings provide insight into the influence of physical cues on cell phenotype maintenance and could help improve understanding of cartilage‐related pathologies such as osteoarthritis.

Exploring Metalloprotease from Dunaliella sp.: Production, Regulation, and Structural Insight

A green microalgal strain, identified as Dunaliella sp., was isolated from the Tunisian southern region. The enhancement of its protein and protease production was performed through culture condition optimization using the response surface methodology. The optimal conditions for protein and protease production were found to be, respectively, (i) NaCl concentrations of 135 and 45.55 g/L, (ii) NaHCO3 concentrations of 0.5 and 1.5 g/L, (iii) temperature of 28 °C for both, and (iv) light intensities of 400 and 100 µmol photons/m2/s. The optimization led to an increase in microalgae protein content from 11.98% ± 0.26 to 18.39% ± 0.10 and microalgae proteolytic activity from 7.36 ± 0.74 U/mg to 12.54 ± 0.86 U/mg. Specific focus was attributed to ATP-dependent metalloprotease, namely, FtsH2, which is involved in numerous cellular processes including cell division, cell differentiation, signal transduction, and stress response. Differential expression of the FtsH2 gene under various stress conditions showed that this expression was upregulated in response to salt stress, gibberellic acid, and Indole-3-butyric acid. A 3D modeling demonstrated two possible arrangements where the ATPase ring shows either a perfect six-fold symmetry with an open circular entrance covering the crucial pore residues, or a translocated model triggered by substrate binding inward movement of the aromatic pore residues.

Nostoxanthin Biosynthesis by Sphingomonas Species (COS14-R2): Isolation, Identification, and Optimization of Culture Conditions

Nostoxanthin, a yellow pigment, belongs to the xanthophyll group of carotenoids found in various species of bacteria and cyanobacteria. Several species of Sphingomonas can produce appropriate carotenoids for survive in various environments. This comprise nostoxanthin, a significant carotenoid. The study isolated the Sphingomonas species strain COS14-R2 from the Cosmos bipinnatus and identified it through the whole-genome sequence. The strain consists of a circular chromosome with a length of 3,677,457 base pairs.s ecThe genome consists of three carotenoid biosynthesis genes, specifically crtB (phytoene synthase), crtI (phytoene desaturase), and crtY (Lycopene beta-cyclase), which are involved in the synthesis of nostoxanthin. The strain has a circular, undulated colony morphology with a deep yellow color. It demonstrates optimal growth in liquid media at 25 to 35 °C and exhibits a high tolerance for pH levels between 5 and 11 and requires adequate quantities of carbon and nitrogen. We observed the highest concentration of nostoxanthin was recorded at 35 °C, pH of 7.5, glucose concentration of 40 g L−1, and a yeast extract concentration of 5 g L−1 during dark incubation. The fed-batch fermentation process produced nostoxanthin at a concentration of 217.22 ± 9.60 mg L−1, with a selectivity of 72.32% and a productivity of 2.59 g/L/h. The dry biomass extract was purified using column chromatography. The LC–MS/MS analysis of the purified fraction indicated that the molecular weight of nostoxanthin is 600.5098 m/z. The DPPH assay result of 75.5 ± 0.33% indicates nostoxanthin is highly effective in scavenging free radicals.

Navigating stem cell culture: insights, techniques, challenges, and prospects.

Stem cell research holds huge promise for regenerative medicine and disease modeling, making the understanding and optimization of stem cell culture a critical aspect of advancing these therapeutic applications. This comprehensive review provides an in-depth overview of stem cell culture, including general information, contemporary techniques, encountered problems, and future perspectives. The article begins by explaining the fundamental characteristics of various stem cell types, elucidating the importance of proper culture conditions in maintaining pluripotency or lineage commitment. A detailed exploration of established culture techniques sheds light on the evolving landscape of stem cell culture methodologies. Common challenges such as genetic stability, heterogeneity, and differentiation efficiency are thoroughly discussed, with insights into cutting-edge strategies and technologies aimed at addressing these hurdles. Moreover, the article delves into the impact of substrate materials, culture media components, and biophysical cues on stem cell behavior, emphasizing the intricate interplay between the microenvironment and cell fate decisions. As stem cell research advances, ethical considerations and regulatory frameworks become increasingly important, prompting a critical examination of these aspects in the context of culture practices. Lastly, the article explores emerging perspectives, including the integration of artificial intelligence and machine learning in optimizing culture conditions, and the potential applications of stem cell-derived products in personalized medicine. This comprehensive overview aims to serve as a valuable resource for researchers and clinicians, fostering a deeper understanding of stem cell culture and its key role in advancing regenerative medicine and biomedical research.

In vitro strategy to enhance the production of bioactive polyphenols and caffeoylputrescine in the hairy roots of Physalis peruviana L.

The Rhizobium rhizogene-transformed root culture from Physalis peruviana L. (P. peruviana) may be a promising and novel source of valuable phenolics, including caffeoylputrescine (CP), which is known for antioxidant, antidiabetic, insect-resistant, disease-resistant, and neuroprotective properties. In this study, to improve the production efficiency of phytochemical components in P. peruviana hairy root cultures, we optimized various culture conditions, including the inoculum size, liquid volume, culture media type, carbon source, sucrose concentration, initial pH, and application of elicitors, to enhance the total phenolic content and CP yield in these hairy root cultures. The findings indicate that the use of sucrose as carbon source resulted in the highest biomass (13.28 g DW/L), total phenolic content (6.26 mg/g), and CP yield (2.40 mg/L). The White medium excelled in enhancing the total phenolic content (9.35 mg/g), whereas the B5 medium was most effective for the biomass (13.38 g DW/L) and CP yield (6.30 mg/L). A sucrose concentration of 5% was best for the biomass (18.40 g DW/L), whereas a sucrose concentration of 4% was ideal for the CP yield. Optimal culture conditions were as follows: an inoculum size of 0.5 g/100 mL, a liquid volume of 100 mL in a 250-mL flask, B5 medium, 4% sucrose, and a pH of 5.5. Among the tested elicitors, methyl jasmonate (MeJA) at 100 µM significantly increased the biomass (21.3 g/L), total phenolic content (23.34 mg/g), and CP yield (141.10 mg/L), which represent 0.96-, 2.12-, and 13.04-fold increases, respectively, over the control after 8 days. The optimized HR culture of P. peruviana provides a promising system to enhance the production of CP for pharmaceutical applications.

Biomass Accumulation, Contaminant Removal, and Settling Performance of Chlorella sp. in Unsterilized and Diluted Anaerobic Digestion Effluent

Microalgae demonstrate significant efficacy in wastewater treatment. Anaerobic digestion effluent (ADE) is regarded as an underutilized resource, abundant in carbon, nitrogen, phosphorus, and other nutrients; however, the presence of inhibitory factors restricts microalgal growth, thereby preventing its direct treatment via microalgae. The purpose of this study was to dilute ADE using various dilution media and subsequently cultivate Chlorella sp. to identify optimal culture conditions that enhance microalgal biomass and water quality. The effects of various dilution conditions were assessed by evaluating the biomass, sedimentation properties, and nutrient removal efficiencies of microalgae. The results demonstrate that microalgal biomass increases as the dilution ratio increased. The microalgae biomass in the treatments diluted with simulated wastewater was significantly higher than that with deionized water, but their effluent quality failed to meet discharge standards. The treatment diluted with deionized water for 10 times exhibited abundant microbial biomass with strong antioxidant properties. The corresponding total phosphorus concentration in the effluent (6.96 mg/L) adhered to emission limits under the Livestock and Poultry Industry Pollutant Emission Standards (8 mg/L), while ammonia nitrogen concentration (90 mg/L) was near compliance (80 mg/L). The corresponding microbial biomass, with a sludge volume index (SVI30) of 72.72 mL/g, can be recovered economically and efficiently by simple precipitation. Its high protein (52.07%) and carbohydrate (27.05%) content, coupled with low ash (10.75%), makes it a promising candidate for animal feed and fermentation. This study will aid in understanding microalgal growth in unsterilized ADE and establish a theoretical foundation for cost-effective ADE purification and microalgal biomass production.

Improved exopolysaccharide production by Lactiplantibacillus plantarum Z-1 under hydrogen peroxide stress and its physicochemical properties

In this study, a strain with good exopolysaccharide (EPS)-producing ability was isolated from the fermented Benincasa hispida and identified as Lactiplantibacillus plantarum Z-1. Its EPS production was further improved by H2O2 stress under optimized culture conditions, increasing from 180 ± 0.45 mg/L to 409.52 ± 2.16 mg/L. Purification of EPS with DEAE-52 and subsequent Sephadex G-100 column chromatography provided three fractions, namely, EPS-0, EPS-1 and EPS-3, respectively. The molecular weight of EPS, EPS-0, EPS-1 and EPS-3 were 85.4, 25.7, 131.88 and 93.2 kDa, respectively. EPS, EPS-1 and EPS-3 were mainly composed of glucose, rhamnose, arabinose and galactose with molar ratios of 1:0.544:0.211:0.281, 1:1.279:0.807:0.704, and 1:1.459:0.759:0.75, respectively, along with small proportions of fucose, mannose and xylose. EPS-0 was composed of glucose, arabinose, galactose and xylose, with molar ratios of 1:0.618:0.206:0.275. The structural analysis indicated that EPS-3 was mainly consisted of (1,2,4)-β-Rhap, (1,2,3)-β-Araf, (1,4)-β-Galp, T-α-Glcp units. The three purified fractions showed typical characteristics of non-Newtonian fluids and good viscoelasticity. Congo red test revealed that irregular triple-helical conformation existed in EPS and EPS-3. These physicochemical properties of EPSs make them a potential candidate for the use as a health-beneficial food additive in the food processing industry.

Biological Properties of S. warneri KYS-164 Isolated from Kefir Grains.

Staphylococcus worderi KYS-164, isolated from homemade Tibetan kefir grains, produces bacteriocin-like inhibitory substances (BLIS), which are peptides with antimicrobial properties, but have not been fully characterized. The research on BLIS will lay the foundation for mining new bacteriocins. In this study, the optimal culture conditions for the production of highly active BLIS were found to be incubation at 30 °C and 120 rpm, and the most effective extraction method was ammonium sulfate precipitation (ASP) using ammonium sulfate at 80% saturation. The postantibiotic effect (PAE) of BLIS on Staphylococcus aureus CICC 10384 is significant, with a 4 × MIC BLIS concentration able to prolong the PAE to 2.39 h. BLIS has excellent biosafety, with no deleterious effects observed at 8 × MIC concentration. Gas chromatography-ion mobility spectrometry (GC-IMS) was used to analyze the volatile compounds synthesized by Staphylococcus warneri KYS-164 during its growth. Hydroxycitronellal, ethyl pyruvate, and α-pinene were found to be unique substances produced by this strain, which can provide fresh, refreshing floral and fruity aromas as well as strong pine and resinous aromas in the process of kefir grain fermentation of milk. Analysis of the S. warneri KYS-164 genome provided insights into the major metabolic pathways in which genes expressed in this strain are involved. This study represents the first isolation of S. warneri KYS-164 from kefir grains prepared by Tibetan families, and provides a comprehensive analysis of its physicochemical properties. This research provides a solid foundation for better understanding and utilization of S. warneri KYS-164.

Design of an Innovative Method for Measuring the Contractile Behavior of Engineered Tissues.

Hypertrophic scarring is a common complication in severely burned patients who undergo autologous skin grafting. Meshed skin grafts tend to contract during wound healing, increasing the risk of pathological scarring. Although various technologies have been used to study cellular contraction, current methods for measuring contractile forces at the tissue level are limited and do not replicate the complexity of native tissues. Self-assembled skin substitutes (SASSs) were developed at the "Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX" and are used as permanent full-thickness skin grafts. The autologous skin substitutes are produced using the self-assembly method, allowing the cultured cells to produce their extracellular matrix leading to a tissue-engineered substitute resembling the native skin. The level of contraction of the SASSs during the fabrication process is patient-dependent. Thus, because of its architecture and composition, SASS is an interesting model to study skin contraction in vitro. Unfortunately, standard measurement methods are unsuited for SASS contraction assessment, mainly due to incompatibilities between the SASS manufacturing process and the current contraction force measurement methods. Here, we present an innovative contraction measurement method specifically designed to quantify the contractile behavior of tissue-engineered substitutes, without disrupting the protocol of production. The method uses C-shape anchoring frames that close at different speeds and magnitudes according to the tissue contractile behavior. A finite element analysis model is then used to associate the frame deformation to a contractile force amplitude. This article shows that the method can be used to measure the contraction force of tissues produced with cells displaying different contractile properties, such as primary skin fibroblasts and myofibroblasts. It can also be used to study the effects of cell culture conditions on tissue contraction, such as serum concentration. This protocol can be easily and affordably applied and tuned to many regenerative medicine applications or contraction-related pathological studies. Impact Statement The protocol presented in this article is a new and simple method to quantify contraction forces present in tissue-engineered substitutes. Using finite element analysis, it allows for the measurement of a contraction force rather than a surface reduction as usually provided by other tissue contraction measurement methods. The results shown are in correlation with the current literature relevant to tissue contraction. It can be easily implemented, and hence, this method will open up new avenues to study tissue contraction of living substitutes engineered with various cell types and to optimize culture conditions.

A New Enzyme for Biodiesel Production and Food Applications: Lipase of Bacillus megateriumF25 Isolated From an Aquatic Insect Rhantus suturalis

ABSTRACTThis study aimed to isolate, purify, and characterize a lipase from the gut symbiont Bacillus megaterium F25 (GenBank accession: MF597792) of the aquatic insect Rhantus suturalis, with a focus on its potential applications in biodiesel and food industries. Under optimized culture conditions, B. megaterium F25 could produce 583 U/L of lipase in shaking flask culture. The purified lipase (PL) exhibited a specific activity with 113.89 U/mg, and its molecular weight was determined as 34 kDa. The activity of PL was enhanced by methanol, ethanol, Tween‐80, Triton X‐100, Ca2+, and Mg2+, while β‐mercaptoethanol, EDTA, SDS, Fe2+, Mn2+, and Cu2+ were inhibitory. PL showed optimal activity and stability at neutral and slightly acidic pHs, as well as in a temperature range of 20°C–30°C. PL displayed strong hydrolytic activity toward plant oils and animal fats, indicating its potency for both the food industry and the remediation of oil‐contaminated environments. When tested as a catalyst, PL provided biodiesel production with a transesterification yield of 86.8% under optimized conditions (36 h reaction time, 4 mL enzyme solution, 30°C, pH 7.0, and waste cooking oil:methanol ratio of 10 mL/40 mL). This is the first report on the lipase‐producing potential of gut microbial symbionts of aquatic insects. Furthermore, B. megaterium lipase was tested for the first time as a biocatalyst for biodiesel production.

Ultraviolet-diethyl sulfate composite mutagenesis of Rhodosporidium toruloides and culture optimization to improve carotenoid production

Rhodosporidium toruloides is a carotenoid-producing oleaginous yeast, and its carotenoids are of considerable value to food, chemical, and medical industries. In this study, to improve carotenoid production capacity, the parent R. toruloides strain (CK) was compound mutagenized using ultraviolet-diethyl sulfate (UV-DES), and cultivation conditions were simultaneously optimized to enhance the carotenoid synthesis ability of the mutant strain (M5). The carotenoid content was significantly increased following UV-DES compound mutagenesis (CK = 249.34 μg/g, M5 = 535.28 μg/g). The carotenoid content of M5 was increased to 763.85 μg/g under optimal culture conditions. In addition, mutagenesis altered the carotenoid components, and torulene and torularhodin levels were also increased following mutation, leading to changes in antioxidant capacity. Real-time quantitative PCR results showed that expression of six genes (CAR1, CAR2, CrtE, CrtI, ME1, HMG1) related to carotenoid accumulation were significantly upregulated by UV-DES composite mutagenesis. In conclusion, combined UV-DES mutagenesis and optimal culture conditions promoted the biosynthesis of carotenoids in R. toruloides.

Batch cultivation of Chlorella vulgaris and simultaneous treatment of restaurant wastewater

BackgroundThe rapid growth of restaurants due to the changing lifestyle has imposed unnecessary impacts on environmental sustainability following an increased generation of restaurant wastewater (RWW). RWW contains alarming concentrations of fats, oils, and greases (FOG), chemical oxygen demand (COD), biochemical oxygen demand (BOD), and nutrients, including nitrogen and phosphorus. Microalgae are known to be able to treat wastewater and provide simultaneous production of biomass and other valuable metabolites (e.g., lipids, proteins, and carotenoids). Numerous studies have been reported on treating various types of wastewater using microalgae. However, studies still need to be reported on treating RWW using microalgae collected from grease traps. MethodsThis research aims to determine the potential of the freshwater microalgae Chlorella vulgaris (Chlorella vulgaris) in treating RWW-containing pollutants (COD, BOD, FOG) and nutrients (TN, TP, AN, K) via optimal autotrophic cultivation conditions (i.e., pH, temperature, light intensity, and aeration). Significant FindingsThe conditions for the batch scale cultivation of Chlorella vulgaris opted as an autotrophic mode with a temperature of 25 °C, aeration of 3 litres per minute supplemented with 3 % CO2 (v/v), and an irradiance range of 80–150 μmol/m2/sec. Maximum specific growth rate and biomass productivity achieved were 0.14 day-1 and 42 mg/l/d, respectively. The maximum pollutant removal efficiency was 98 % for COD, 98.5 % for BOD, 96.8 % for FOG. While the nutrient uptake achieved was 99.7 % for total nitrogen (TN), 99.9 % for ammoniacal nitrogen (AN), 99.9 % for total phosphorus (TP), and 97.8 % for potassium (K). Therefore, this study shall provide an alternative potential solution by proposing treatment using microalgae and cultivating it with RWW. No study has been reported to date using freshwater microalgae Chlorella vulgaris to evaluate its potential in removing pollutants, nutrients, and FOG in RWW collected from GGI. The removal efficiencies indicated that the RWW acclimatised well with Chlorella vulgaris, thus providing an environmentally sustainable and economically viable treatment method.