Cell Biomass Research Articles

Antimicrobial and antibiofilm activity of human recombinant H1 histones against bacterial infections.

Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombinant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.IMPORTANCEThe constant increase of multidrug-resistant bacteria is a critical global concern. The inefficacy of current therapies to treat bacterial infections is attributed to multiple mechanisms of resistance, including the capacity to form biofilms. Therefore, the identification of novel and safe therapeutic strategies is imperative. This study confirms the antimicrobial potential of three histone H1 subtypes against both Gram-negative and Gram-positive bacteria. Furthermore, histones H1.0 and H1.4 demonstrated in vivo efficacy without associated toxicity in an acute infection model of Pseudomonas aeruginosa PAO1 in Galleria mellonella larvae. The bactericidal effect of these proteins also resulted in biomass reduction of P. aeruginosa PAO1 biofilms. Given the clinical significance of this opportunistic pathogen, our research provides a comprehensive initial evaluation of the efficacy, toxicity, and mechanism of action of a potential new therapeutic approach against acute and chronic bacterial infections.

Enhancing the volatile organic compound and biomass production by three biocontrol potential bacteria in corn steep liquor growth medium and development of cell freeze-drying process.

This study investigates the traits of three plant growth-promoting (PGP) and antagonistic bacteria, Pseudomonas protegens MP12, Bacillus sp. 3R4, and Bacillus sp. T22, to assess their potential application as biocontrol agents by using the ecofriendly and low-cost substrate Corn Steep Liquor (CSL) medium. Analyses of antagonism through volatile organic compounds (VOCs) production, biofilm formation, and growth performance were carried out. Dual antagonism assay showed that all strains displayed significant antagonistic activity against Botrytis cinerea through VOCs. Gas chromatography demonstrated that strains in the CSL exhibited higher VOCs production than Nutrient medium. Moreover, enhanced biofilm formation analysed by Calgary Biofilm Device, growth and biomass were noted in CSL cultures. Pseudomonas protegens MP12, which showed higher cell concentration and biomass yield, was selected for freeze-drying treatments. Storage cell viability assays evidenced that it can be effectively preserved for nearly 7 months at 4°C. The results here obtained showed that CLS medium enhance VOCs production, biofilm formation, growth and biomass of the antagonistic bacteria of the three strains. Eventually, the more effective strain P. protegens MP12 can be stored for nearly 7 months at 4°C.

Molecular mechanism of boosted hydrogen production by Thermoanaerobacterium thermosaccharolyticum with biochar revealed by transcriptome analysis

Although numerous studies have reported the enhancement of hydrogen production by biochar in dark fermentation, the molecular mechanisms underlying this enhancement remain poorly understood. This study investigated the role of biochar in boosting hydrogen production by Thermoanaerobacterium thermosaccharolyticum MJ2. The results showed that BC600 addition led to a 290.7% increase in hydrogen production and a 67.1% improvement in hydrogen yield, with a significant increase in cell biomass, 74% of which was immobilized on biochar. Transcriptome analysis revealed the upregulation of genes coding for hydrogen-producing ech hydrogenases and the downregulation of hnd hydrogenase genes involved in hydrogen consumption. Additionally, gene expression related to cell immobilization, nitrogen fixation for hydrogen production were also enhanced, while genes associated with glycolysis showed an overall downregulation. For the key genes of interest, BC600 had a more profound effect on their expression levels in immobilized cells compared to suspended cells within the same system. This study provides valuable insights into how biochar regulates microbial metabolism and gene expression to enhance hydrogen production, aiding in the development of more effective biochar application strategies for biohydrogen production.

Enhancing the production and immobilization of cell‐bound lipase from yeast‐like fungus Magnusiomyces capitatusA4C for sustainable biodiesel production in a packed bed reactor

AbstractMagnusiomyces capitatus A4C, a mycelium‐forming and lipase‐producing yeast‐like fungus, was employed in a five‐level factorial design to optimize the collective and interactive influences of carbon, nitrogen and emulsifying sources and their possible effects on cell‐bound lipase (CBL) and cell biomass production. The cell culture of M. capitatus A4C was incubated along with biomass support particles (BSPs) to immobilize the enzyme while anchoring CBL on their surfaces. Among the BSPs tested, CBL immobilized on loofah sponge under optimized conditions showed a substantial hydrolytic activity of 12.7 U mL−1 and a cell‐loading capacity of 0.61 g g−1 of BSPs. Immobilized CBL was applied for biodiesel production via transesterification and esterification. The conversion percentage of triacylglycerides was approximately 100% at 24 h with the addition of water at 1:1 (v/v). The conversion of oleic acid into biodiesel via esterification was 100% at 48 h in the presence of 15% (v/v) isooctane. Further, biodiesel production was scaled up using a packed bed reactor. The batch production of biodiesel in a packed bed reactor through transesterification was 96.2%, with a circulation flow rate of 5.5 mL min−1 for 18 h. On the other hand, oleic acid conversion into biodiesel via esterification was 99.5%, with a circulation flow rate of 5.5 mL min−1 for 24 h. Further investigation revealed that the immobilized biocatalyst exhibited higher stability with esterification (85.3% fatty acid methyl ester) after ten repeated cycles.

Investigating the Potential of River Sediment Bacteria for Trichloroethylene Bioremediation

Trichloroethylene (TCE) is a prevalent groundwater contaminant detected worldwide, and microbes are sensitive indicators and initial responders to these chemical contaminants causing disturbances to their ecosystem. In this study, microbes isolated from San Marcos River sediment were screened for their TCE degradation potential. Among the twelve isolates (SAN1-12), five isolates demonstrated TCE degradation within 5 days at 25 °C and 40 mg/L of TCE concentration in the following order: SAN8 (87.56%), SAN1 (77.31%), SAN2 (76.58%), SAN3 (49.20%), and SAN7 (3.36%). On increasing the TCE concentration to 80 mg/L, the degradation efficiency of these isolates declined, although SAN8 remained the prominent TCE degrader with 75.67% degradation. The prominent TCE-degrading isolates were identified as Aeromonas sp. SAN1, Bacillus sp. SAN2, Gordonia sp. SAN3, and Bacillus proteolyticus SAN8 using 16S rRNA sequencing. The TCE degradation and cell biomass of Bacillus proteolyticus SAN8 were significantly improved when the incubation temperature was increased from 25 °C to 30 °C. However, both slightly acidic and alkaline pH levels, as well as higher TCE concentrations, lowered the efficacy of TCE degradation. Nevertheless, these conditions led to an increase in bacterial cell biomass.

Withaferin A decreases glycolytic reprogramming in breast cancer

Reprogrammed glucose metabolism is considered as the hallmark of cancer with therapeutic implications. Phytocompounds have potential to inhibit cancer metabolism. Here, we tested the ability of Withaferin A (WA), a withanolide derived from Withania somnifera, in modulating cancer metabolism. The assessed effect of WA on aerobic glycolysis in breast cancer cell lines showed that WA decreases the glucose uptake, lactate production and ATP generation by inhibiting the expression of key glycolytic enzymes i.e., GLUT1, HK2 and PKM2. We also identified that WA induced inhibition of cancer glycolysis by targeting c-myc as validated by silencing experiments followed by metabolic readouts. Decreased glycolysis resulted in reduced cell viability, biomass and colony forming ability of breast cancer cells. To further validate our in vitro findings in breast cancer patients, we analyzed 90 metabolic pathways in ~ 2000 breast tumors and observed that glycolysis is the most deregulated pathway in breast tumors. Deregulated glycolysis also predicted poor prognosis in breast cancer patients. In addition, patient data showed correlation between c-myc expression and glycolytic deregulation in breast cancer. Taken together, our results highlight the role of WA in inhibiting breast cancer metabolism via c-myc/glycolysis axis.

Phytoplankton morphological traits and biomass outline community dynamics in a coastal ecosystem (Gulf of Trieste, Adriatic Sea)

AbstractTrait-based ecology has recently gained increasing importance in phytoplankton research. In particular, the taxonomic and morphological traits, such as size and shape of phytoplankton cells, can help to unveil the ecological processes and their drivers in the pelagic domain. Our study aims to shed light on the trophodynamics of phytoplankton communities in a coastal ecosystem in the northern Adriatic Sea (Gulf of Trieste) using data on individual traits such as biomass, size and shape of phytoplankton taxa during a one-year study. The phytoplankton parameters were investigated at the levels of the whole community, groups, and individual cells, analysing also the probability distributions of biomass and size of the latter level. The results showed good agreement between abundance and biomass data, as well as individual size and biomass with differences partly explained by cell shapes. We have emphasized the role of the local freshwater source in bottom-up control, alternating with top-down control of phytoplankton dynamics through taxonomic and morphological diversity. The predominant bimodal and non-power law distribution, especially during and around the biomass peaks, confirmed the importance of nano- and microphytoplankton size classes and the role of blooms in destabilizing the trophic webs. We suggest that the analyses of distribution types of individual cell size and biomass can be appropriate to spot ecological processes driving to unconstrained phytoplankton proliferation or to periods of trophic web stability.

Morphological, cytogenetic, and phytochemical estimation of Lychnis flos-cuculi L. cell cultures: can cell biomass be a source of bioactive compounds?

The ragged robin (Lychnis flos-cuculi L.) from the Caryophyllaceae family contains flavonoids, phenolic acids, flavonoids, triterpenoid saponins, and ecdysteroids, making it potential for biological and medicinal application. Plant cell culture technologies could be beneficial for obtaining low-cost biomass sources with valuable phytochemicals sustainably. The current study reports the induction and proliferation of ragged robin cell culture and evaluation of various plant growth regulators (PGRs) impact on the secondary metabolites profile using UPLC-HRMS/MS. Callus was initiated from different explants on the Murashige and Skoog medium with various combinations and concentrations of PGRs. It was found that prolonged proliferation on MS medium enriched with a combination of 1.0 mg l− 1 2,4-Dichlorophenoxyacetic acid and 0.1 mg l− 1 N6-furfuryl adenine (Kinetin) yields the maximum biomass with 504.05% growth index. The results of flow cytometric analysis suggested no genetic alteration between differentiated and dedifferentiated cultures. Furthermore, the UPLC-HRMS/MS result distinguished 101 significant signals that were manually identified as ecdysteroids (16 metabolites), flavonoids (11 metabolites), phenolic acids (10 metabolites), triterpene saponins and sapogenins (53 metabolites), and some primary metabolites. Our study demonstrates that the cell culture of L. flos-cuculi displays significant differences in its phytochemical makeup to plants cultivated in vitro. Moreover, the type of growth regulators used for callus cultures was identified to cause substantial shifts in the identified metabolites.Graphical

Prebiotic activity of enzymatically modified pea peel dietary fiber: An in vitro study

The study examined the prebiotic index of enzymatically modified insoluble dietary fiber from pea peel by using three probiotic bacterias; Lactobacillus rhamnosus ATCC 7469, Lactobacillus sakei ATCC 15521, Lactobacillus plantarum ATCC 8014 and one pathogenic bacteria; Escherichia coli ATCC 4157. A positive value for prebiotic potential was determined which was greater than inulin, unmodified dietary fiber, and standard glucose. The 30 h of cultivation, the cell density obtained from Lactobacillus rhamnosus ATCC 7469 culture conducted in MRS was 7.45 ± 0.08 log CFU/mL in glucose medium and 7.37 ± 0.07 log CFU/mL in dietary fiber medium. Growth kinetics analysis showed a positive effect for Lactobacillus spp. in DF medium. DF could boost cell biomass and relative growth rate of probiotic bacteria. Thus, the microbiome of the colon facilitates dietary fiber fermentation by bacteria shown in in vitro simulated gastrointestinal model.

Effects of different trophic conditions on total fatty acids, amino acids, pigment and gene expression profiles in Euglena gracilis.

Euglena gracilis is a unique microalga that lacks a cell wall and is able to grow under different trophic culture conditions. In this study, cell growth, biomass production, and changes in the ultrastructure of E. gracilis cells cultivated photoautotrophically, mixotrophically, and under sequential-heterotrophy-photoinduction (SHP) were assessed. Mixotrophy induced the highest cell growth and biomass productivity (6.27 ± 0.59mg/L/d) in E. gracilis, while the highest content of fatty acids, 2.69 ± 0.04% of dry cell weight (DCW) and amino acids, 38.16 ± 0.08% of DCW was obtained under SHP condition. E. gracilis also accumulated significantly higher saturated fatty acids and lower unsaturated fatty acids when cultivated under SHP condition. Transcriptomic analysis showed that the expression of photosynthetic genes (PsbA, PsbC, F-type ATPase alpha and beta) was lower, carbohydrate and protein synthetic genes (glnA, alg14 and fba) were expressed higher in SHP-culture cells when compared to other groups. Different trophic conditions also induced changes in the cell ultrastructure, where paramylon and starch granules were more abundant in SHP-cultured cells. The findings generated in this study illustrated that aerobic SHP cultivation of E. gracilis possesses great potential in human and animal feed applications.

Potato and dairy industry side streams as feedstock for fungal and plant cell cultures

Utilization of industrial side streams as nutrient source for cellular agriculture is a promising option to improve the sustainability of the production processes. The aim of the study was to evaluate usability of two liquid food industry side streams, potato cell fluid and acid whey, as nutrient source for production of food ingredients with plant cell cultures (arctic bramble (Rubus arcticus L.) and tobacco BY-2 (Nicotiana tabacum L.)) and filamentous fungi (Paecilomyces variotii, Rhizopus oligosporus and Trichoderma reesei). The side streams were found to contain various sugars and other potential nutrients suitable for the studied organisms. The side streams were used as a sole nutrient source (fungi) or as a replacement of selected nutrients in the growth media (fungi and plant cells) in flask scale cultivations. Acid whey was successfully used as a growth medium for filamentous fungi, but it inhibited plant cell growth presumably due to high organic acid content (14 g/l). Potato side stream was found suitable as a fungal and plant cell growth media supplement, and it was used together with glucose for filamentous fungi, or as a partial replacer of macronutrients for plant cells, in bioreactor cultivations yielding high fungal (up to 36 g/l dry weight) and good plant cell (9.5 g/l dry weight) biomass production. Analyses of the produced biomasses revealed good nutritional value in terms of amino acid (17–27 % of dry matter) and dietary fiber (23–30 % of dry matter) contents, giving good premises for utilization in human nutrition.

Plankton and contour algal communities in the Ukrainian section of the Western Bug River and its tributaries

The paper deals with spatial dynamics of cell count, biomass, dominant species complexes, information diversity of planktonic and contour algal communities in the Western Bug River and its tributaries. The trophic state and water quality of the rivers under study have been assessed. The spatial heterogeneity of phytoplankton, microphytobenthos and phytoperiphyton brought about high cell count and biomass values during the low-water summer-autumn season. The cell count and biomass have been shown to increase from the upper reaches downstream, correlating with the river bed width. High quantitative diversity of algal communities was due to Bacillariophyta dominance. Cyanobacteria and Chlorophyta were recorded as subdominants. The trophic state of the aquatic ecosystems under study varied between oligotrophic and eutrophic. The Shannon’s index made up 1.58–4.62 bit/cell and 1.60–4.40 bit/mg. The trophic state and information diversity assessed according to contour algal communities were higher than according to phytoplankton. This is indicative of the clear water regime forming in the rivers under study during the low-water summer-autumn season. The obtained findings differ from the data, published earlier for the Kaniv Water Reservoir, where the primary role belonged to phytoplankton. The spatial heterogeneity of algal communities is related to the dominant complexes structure. As a rule, these are monodominant Bacillariophyta complexes or oligodominant Bacillariophyta – Cyanobacteria or Bacillariophyta – Chlorophyta complexes. The water quality assessment according to the abiotic variables and saprobiologic characteristics has shown that the modal classes of water quality are the 2nd–3rd classes (clean waters – satisfactory clean waters). Therefore, the water quality of the Ukrainian section of the Western Bug River and its tributaries does not pose any substantial hazard to the adjacent European countries.

Endophyte-based fungal elicitors for enhanced production of valepotriates and sesquiterpenoids in leaf cell suspension cultures of Valeriana jatamansi Jones.

The immense therapeutic value of Valeriana jatamansi is attributed to the presence of bioactive secondary metabolites (valepotriates and sesquiterpenoids). Its over-exploitation in wild habitats resulted in extensive depletion, necessitating alternative approaches to produce its therapeutic metabolites. This study sought to assess the ability of endophytes of V. jatamansi to boost the biosynthesis of secondary metabolites in the leaf-cell suspension (LCS) culture of V. jatamansi. A total of 11 fungal endophytes were isolated from the rhizomes of V. jatamansi. Isolated endophytes were found to belong to phylum Ascomycota, Basidiomycota, and Mucoromycota. Supplementation of extracts of endophyte Phaeosphaeriaceae sp. VRzFB, Mucor griseocyanus VRzFD, Penicillium raistrickii VRzFK, and Penicillium sajarovii VRzFL in the LCS culture of V. jatamansi increased the fresh cell biomass by 19.6%-39.1% and dry cell biomass by 23.4%-37.8%. Most of the endophytes' extract could increase the content of valepotriates (26.5%-76.5% valtrate and 40.5%-77.9% acevaltrate) and sesquiterpenoids (19.9%-61.1% hydroxyl valerenic acid) in LCS culture. However, only two endophytes, Irpex lacteus VRzFI and Fusarium oxysporum VRzFF, could increase the sesquiterpenoids acetoxy valerenic acid (36.9%-55.3%). In contrast, some endophytes' extracts caused negative or no significant effect on the cell biomass and targeted metabolites. Increased secondary metabolites were corroborated with increased expression of iridoid biosynthesis genes in LCS culture. Production of H2O2 and lipid peroxidation was also varied with different endophytes indicating the modulation of cellular oxidative stress due to endophyte elicitors. The results suggest the distinct effect of different fungal endophytes-extract on LCS culture, and endophytes can serve as biotic elicitors for increasing the secondary metabolite production in plant in vitro systems.

Integration of induction, system optimization and genetic transformation in Veratrum californicum var. vitro cultures to enhance the production of cyclopamine and veratramine

Cyclopamine, a compound found in wild Veratrum has shown promising potential as a lead anti-cancer drug by effectively blocking cancer signaling pathways. However, its complex chemical structure poses challenges for artificial synthesis, thus limiting its supply and downstream drug production. This study comprehensively utilizes induction, system optimization, and transgenic technologies to establish an efficient suspension culture system for the high-yield production of cyclopamine and its precursor, veratramine. Experimental results demonstrate that methyl jasmonate (MeJA) effectively promotes the content of veratramine and cyclopamine in Veratrum californicum var. callus tissue, while yeast extract (YE) addition significantly increases cell biomass. The total content of veratramine and cyclopamine reached 0.0638 mg after synergistic treatment of suspension system with these two elicitors. And the content of the two substances was further increased to 0.0827 mg after the optimization by response surface methodology. Subsequently, a genetic transformation system for V. californicum callus was established and a crucial enzyme gene VnOSC1, involved in the steroidal alkaloid biosynthesis pathway, was screened and identified for genetic transformation. Combined suspension culture and synergistic induction system, the total content of the two substances in transgenic suspension system was further increased to 0.1228 mg, representing a 276.69% improvement compared to the initial culture system. This study proposes a complete and effective genetic transformation and cultivation scheme for V. californicum tissue cells, achieving milligram-level production of the anticancer agent cyclopamine and its direct precursor veratramine for the first time. It provides a theoretical basis for the industrial-scale production of these substances.

The adaptation of lichen symbiosis to desert saline-alkali stress depends more on their symbiotic algae.

Soil salinization is a major environmental threat to the entire terrestrial ecosystem. Lichens arose from the symbiosis of fungi and algae or cyanobacteria. They have a high tolerance to various extreme environments, including adaptation to saline-alkali habitats. Thus, lichens are pioneer species on saline-alkali soil. However, the separate resilience of the two symbiotic partners under saline-alkali conditions remains insufficiently understood. In this study, two representative symbiotic algae, Diplosphaera chodatii and Trebouxia jamesii, were studied for their physiological response to the saline-alkali stress by adjusting different concentrations of NaHCO3, together with their respective symbiotic fungi Endocarpon pusillum (terricolous lichen) and Umbilicaria muhlenbergii (saxicolous lichen). The results indicate that cell growth rate and biomass in all four cultures decreased in alkali-alkaline substrate, while cellular activities and ultrastructure were affected to a distinct extent. Compared with the symbiotic fungi, the algae were found to be more active in coordinating oxidative stress and lipid peroxidation damage under the saline-alkali stress. The antioxidant system of the alga was especially shown as a key adaptive trait and it provides an important strategy for species survival and persistence in arid saline-alkali desert. The specific survival ability of the lichen symbiosis relies on the stress resilience advantages of the symbiotic partners in combination. Our study provided new insights into understanding the adaptation of lichen symbiosis to desert saline-alkali soil, and the potential of lichen symbiotic algae in the future desert ecological restoration.

Evaluation of Pan-RAF Inhibitor LY3009120 on Human Uveal Melanoma Cell Line 92-1.

Uveal melanoma (UM) represents a prevailing primary intraocular malignancy, with a limited median overall survival among metastatic patients, and most tumors lack RAF/RAS mutations. The pan-RAF inhibitor LY3009120 has demonstrated valuable anti-tumor effects in a wide range of RAF/RASmut and wild-type (WT) tumor models. This study aimed to evaluate the antitumor effect of LY3009120 on 92-1 UM cell line. The effect of the pan-RAF inhibitor LY3009120 on cell proliferation, metabolic activity, biomass, early and late apoptosis/necrosis, and morphology was characterized in vitro (0.1-5 μM for 48 h/72 h). Furthermore, targeted panel sequencing was used to characterize the mutational landscape of the human 92-1 UM cell line. LY3009120 showed a significant concentration-dependent anti-proliferative effect on 92-1 cells. Cell proliferation and viability were significantly reduced at the lowest effective concentration of 0.5 μM (at 48 and 72 h, p<0.001). Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in 92-1 cells at 5 μM. Except for TP53, NGS showed that all 49 additional analysed genes (Oncomine myeloid panel) of 92-1 were wild-type, including BRAF, NRAS, and KRAS. The pan-RAF inhibitor LY3009120 demonstrated a significant anti-tumor effect on human UM cell line 92-1 independent of the molecular BRAF and RAS mutational status.

Overexpression of native carbonic anhydrases increases carbon conversion efficiency in the methanotrophic biocatalyst Methylococcus capsulatus Bath.

Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH4 as a carbon and energy source. Evidence suggests that some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH4 and CO2 metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show that M. capsulatus expresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO2-dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO2 limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles in M. capsulatus metabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO2 evolution from CH4-consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH4 to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH4 biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH4 into CO2 and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show that M. capsulatus carbonic anhydrases are critical for this bacterium to optimally utilize CO2. We developed an engineered strain with improved CO2 utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH4 conversion bioprocesses.

A protocol for the development and maintenance of Coffea arabica (L.) cell suspension cultures

Coffea spp. are remarkable sources of phytochemicals, but the lack of a well-defined culture medium aimed at the induction of non-embryogenic and friable callus hampers the establishment of plant cell suspension cultures for large-scale production of valuable compounds. In this paper, we describe a one-medium protocol suitable to obtain both callus and cell suspension cultures from leaves of two elite cultivars of C. arabica. The protocol was developed through an iterative process involving the determination of the best concentration of auxin and cytokinin, their optimal ratio, as well as the most effective molecule of either hormone class. Young leaves were found to be a good and easy-to-use explant source for callus induction and proliferation, provided that a cytokinin was present in association with a chlorinated auxin in a full strength, semi-solid MS medium. The best results were obtained by hormone concentration and combination of 1 mg/L of both kinetin and 2,4,5-trichlorophenoxyacetic acid. The same ratio of these growth regulators was conveniently used for the development and stabilization of cell suspension cultures in liquid MS medium. When grown in darkness, stabilized suspension cultures showed a fine and homogeneous texture, with a 10-fold biomass increase within 25 days and a cell viability > 90%. In addition, the phytochemical profile revealed the presence of the most widely studied coffee compounds. The protocol can be applied to obtain adequate amounts of cell biomass for use in physiological studies concerning the production of secondary metabolites.

Optimization of Lipid Production of Rhodosporidium toruloides from Various Substrates

Background and Aim: In recent years, bioenergy has received a lot of attention from a technical point of view due to its ability to use reversible resources and its applicability worldwide. Biodiesel is a non-toxic, safe, renewable, and degradable fuel that is obtained from natural sources, such as vegetable oils, food waste oils, animal fats, algae, and microorganisms. This study aimed to optimize the lipid production of Rhodosporidium toruloides as a biofuel from different substrates. Materials and Methods: In this experimental laboratory study, after lipid extraction from Rhodosporidium toruloides CECT1137, cell biomass, nitrogen, and sugar consumption were measured according to the Bly and Dyer method. Then, using gas chromatography, the produced lipid compounds were identified, and lipid production was optimized by the one-factor method (type of nitrogen source and KH2PO4) and the Taguchi method (carbon and nitrogen source, temperature, pH, centrifuge, and time). Finally, the feasibility of mutation was investigated using UV to optimize lipid production. Results: The amount of lipid produced by Rhodosporidium toruloides was 4.95 g/l, which, after optimization by the Taguchi method, reached 8.54 g/l. Cell biomass, residual nitrogen, and consumed sugar were measured as 14.05, 0.19, and 28.85 g/l, respectively. Optimal production conditions were estimated by Taguchi, including 80 g/l carbon source, 1 g/l nitrogen source, 25 ° C, pH = 5.5, centrifuge at 180 rpm, and 96 hours. Most lipids produced were oleic acid and palmitic acid. Lipid production increased after UV exposure. Conclusion: The results showed that Rhodosporidium toruloides can accumulate a significant amount of lipid in its cells and can be a good alternative to plants and other sources of fuel production.

Development of a Zn-Based Single-Atom Nanozyme for Efficient Hydrolysis of Glycosidic Bonds.

Hydrolytic enzymes are essential components in second-generation biofuel technology and food fermentation processes. Nanozymes show promise for large-scale industrial applications as replacements for natural enzymes due to their distinct advantages. However, there remains a research gap concerning glycosidase nanozymes. In this study, a Zn-based single-atom nanozyme (ZnN4-900) is developed for efficient glycosidic bond hydrolysis in an aqueous solution. The planar structure of the class-porphyrin N4 material approximatively mimicked the catalytic centers of natural enzymes, facilitating oxidase-like (OXD-like)activity and promoting glycosidic bond cleavage. Theoretical calculations show that the Zn site can act as Lewis acids, attacking the C─O bond in glycosidic bonds. Additionally, ZnN4-900 has the ability to degrade starch and produce reducing sugars that increased yeast cell biomass by 32.86% and ethanol production by 14.56%. This catalyst held promising potential for enhancing processes in ethanol brewing and starch degradation industries.