Early Exponential Phase Research Articles

Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli.

5-Aminolevulinic acid (ALA) is an important metabolic intermediate compound with high value and has recently been used in agriculture and medicine. In this study, we have constructed six recombinant Escherichia coli (E. coli) strains that are involved in pET system under the regulation of the T7 promoter and LacI to express codon-optimized hemA gene from Rhodobacter capsulatus (RchemA) for ALA production via the C4 pathway. Due to codon optimization, hemA has a high transcriptional level; however, most RcHemA proteins were formed as inclusion body. To improve expression in soluble form, the vector with TrxA fusion tag was successfully used and co-expressed with partner GroELS as chaperone in another vector. As a result, ALA production increased significantly from 1.21 to 3.67g/L. In addition, optimal ALA production was developed through adjustment of induction time and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, as well as substrate addition conditions. By adopting a two-stage induction strategy, the highest ALA reached 5.66g/L when 0.1mM of IPTG was added at early exponential phase (i.e., OD600 was equal to 0.7 to 0.8), while 6g/L of glycine, 2g/L of succinate, and 0.03mM of pyridoxal 5'-phosphate (PLP) were provided in the mid-exponential phase in fermentation.

Pulsed electric field-assisted fermentation of Hanseniaspora sp. yeast isolated from Lebanese apples.

Hanseniaspora sp. yeast was stimulated using pulsed electric field (PEF) during the different fermentation phases. The impact of PEF parameters on the growth rate and substrate consumption was studied. The PEF intensities chosen for this study were mainly in the range of 72-285V cm-1. A PEF treatment chamber was designed for this study with a ratio of 1:50 between the volume of the fermenter and the volume of the chamber. It allows the recycling of the culture medium using a peristaltic pump, and the yeast treatment by PEF during the fermentation. The continuous circulation of the medium allows avoiding the increase of the temperature inside the fermenter, the cell aggregation, as well as the agitation and the scale-up issues that are associated with the PEF treatment of the entire volume in batch mode. The maximal yeast growth rate was obtained using an electric field strength of 285V cm-1 applied during both Lag and early exponential phase, and Log phase. This observation was accompanied by a faster consumption of glucose in the medium during the fermentation. Besides, the sensitivity of Hanseniaspora sp. yeast to PEF treatment was more pronounced during the Lag and early exponential phase than the Log phase. The results obtained exposed the great benefit of stimulating Hanseniaspora sp. yeast using moderate PEF as it reduces the fermentation time along with increasing the biomass concentration.

Influence of the Crc global regulator on substrate uptake rates and the distribution of metabolic fluxes in Pseudomonas putida KT2440 growing in a complete medium.

When the soil bacterium Pseudomonas putida grows in a complete medium, it prioritizes the assimilation of preferred carbon sources, optimizing its metabolism and growth. This regulatory process is orchestrated by the Crc and Hfq proteins. The present work examines the changes that occur in metabolic fluxes when the crc gene is inactivated and cells grow exponentially in LB complete medium. Analyses were performed at three different moments during exponential growth, examining the assimilation rates for the compounds present in LB, changes in the proteome, and the changes in metabolic fluxes predicted by the iJN1411 metabolic model for P. putida KT2440. During the early exponential phase, consumption rates for sugars, many organic acids and most amino acids were higher in a Crc‐null strain than in the wild type, leading to an overflow of the metabolic pathways and the leakage of pyruvate and acetate. These accelerated consumption rates decreased during the mid‐exponential phase, when cells mostly used sugars and alanine. At later times, pyruvate was recovered from the medium and utilized. The higher consumption rates of the Crc‐null strain reduced the growth rate. The lack of the Crc/Hfq regulatory system thus led to unbalanced metabolism with poorly optimized metabolic fluxes.

A Numerical Study of Lean Propane-Air Flame Acceleration at the Early Stages of Burning in Cold and Hot Isothermal Walled Small-Size Tubes

In this study, the problem of lean propane-air premixed flame acceleration from closed to open end during the early stages of burning in small-size tubes with isothermal walls was considered. In particular, the effects of tube radius, slip/non-slip wall conditions, and the wall temperature on the flame propagation and shape were investigated numerically. Five stages of flame propagation are identified: 1) spherical expansion of the flame front; 2) finger shape expansion of the flame front before touching the wall; 3) flame propagation in the tube subjected to flame-wall interactions; 4) transformation of the flame shape into tulip form; 5) conversion of the tulip shape flame to finger. Our results show that the tube radius, wall condition and its temperature significantly affect flame propagation regimes even in the first instance of the flame propagation in the tubes. We find that increasing tube radius has, overall, the effect of increasing the flame propagation speed in isothermal tubes. Also, depending on tube radius and wall condition, the wall temperature can increase or decrease the flame propagation speed in the isothermal tubes. Furthermore, the results demonstrate that imposing either slip or non-slip condition on the tube’s walls impressively affects flame acceleration and its configuration in the early stages. We observe that, unlike flame propagation forms in the tubes with slip walls, the early exponential flame propagation phase in the tubes was generally followed by a linear flame propagation phase in the tubes with a non-slip wall condition. We obtain that flame propagation in tubes with slip wall conditions are more sensitive to variations in tube radius and wall temperature compared to non-slip conditions. We also see that, contrary to the exponential flame propagation phase, increasing the non-slip wall temperature reduces the flame propagation speed in the linear part of the flame propagation, while such an increase in temperature leads to oscillations in the flame propagation speed in the tubes with slip walls.

Continuous nanosecond pulsed electric field treatments foster the upstream performance of Chlorella vulgaris-based biorefinery concepts

Nanosecond pulsed electric field treatment (nsPEF) is an innovative, technology-driven, and resource-efficient approach to foster the upstream performance of microalgae-based biorefinery concepts to transform microalgae into economic more viable raw materials for the biobased industry. A processing window applying three treatments of 100 ns, 5 Hz, and 10 kV cm−1 to industrially relevant phototrophic Chlorella vulgaris in the early exponential growth phase significantly increased biomass yields by up to 17.53 ± 10.46% (p = 3.18 × 10−5). Treatments had limited effects on the carbon and pigment contents, but the protein content was decreased. The longest possible pulse width (100 ns) resulted in the highest biomass yield indicating underlying working mechanisms of enhanced cell proliferation based on intracellular and plasma membrane-related effects. The applicability to eukaryotes and prokaryotes, such as C. vulgaris and cyanobacteria highlights the possible impacts of nsPEF across multiple domains of the biobased industry relying on single-cell-based value-chains.

Metabolomic profiling of CHO fed-batch growth phases at 10, 100, and 1,000 L.

Established bioprocess monitoring is based on quick and reliable methods, including cell count and viability measurement, extracellular metabolite measurement, and the measurement of physicochemical qualities of the cultivation medium. These methods are sufficient for monitoring of process performance, but rarely give insight into the actual physiological states of the cell culture. However, understanding of the latter is essential for optimization of bioprocess development. Our study used LC-MS metabolomics as a tool for additional resolution of bioprocess monitoring and was designed at three bioreactors scales (10 L, 100 L, and 1,000 L) to gain insight into the basal metabolic states of the Chinese hamster ovary (CHO) cell culture during fed-batch. Metabolites characteristics of the four growth stages (early and late exponential phase, stationary phase, and the phase of decline) were identified by multivariate analysis. Enriched metabolic pathways were then established for each growth phase using the CHO metabolic network model. Biomass generation and nucleotide synthesis were enriched in early exponential phase, followed by increased protein production and imbalanced glutathione metabolism in late exponential phase. Glycolysis became downregulated in stationary phase and amino-acid metabolism increased. Phase of culture decline resulted in rise of oxidized glutathione and fatty acid concentrations. Intracellular metabolic profiles of the CHO fed-batch culture were also shown to be consistent with scale and thus demonstrate metabolomic profiling as an informative method to gain physiological insight into the cell culture states during bioprocess regardless of scale.

Proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production

BackgroundRhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids.ResultsRhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose.ConclusionsThe results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.

Transition of Bacterial Diversity and Composition in Tongue Microbiota during the First Two Years of Life.

Newborns are constantly exposed to various microbes from birth; hence, diverse commensal bacteria colonize the oral cavity. However, how or when these bacteria construct a complex and stable ecosystem remains unclear. This prospective cohort study examined the temporal changes in bacterial diversity and composition in tongue microbiota during infancy. We longitudinally collected a total of 464 tongue swab samples from 8 infants (age of <6 months at baseline) for approximately 2 years. We also collected samples from 32 children (aged 0 to 2 years) and 73 adults (aged 20 to 29 years) cross-sectionally as control groups. Bacterial diversities and compositions were determined by 16S rRNA gene sequencing. The tongue bacterial diversity in infancy, measured as the number of observed operational taxonomic units (OTUs), rapidly increased and nearly reached the same level as that in adults by around 80 weeks. The overall tongue bacterial composition in the transitional phase, 80 to 120 weeks, was more similar to that of adults than to that of the early exponential phase (EEP), 10 to 29 weeks, according to analysis of similarities. Dominant OTUs in the EEP corresponding to Streptococcus peroris and Streptococcus lactarius exponentially decreased immediately after EEP, around 30 to 49 weeks, whereas several OTUs corresponding to Granulicatella adiacens, Actinomyces odontolyticus, and Fusobacterium periodonticum reciprocally increased during the same period. These results suggest that a drastic compositional shift of tongue microbiota occurs before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years.IMPORTANCE Evaluating the development of oral microbiota during infancy is important for understanding the subsequent colonization of bacterial species and the process of formation of mature microbiota in the oral cavity. We examined tongue microbiota longitudinally collected from 8 infants and found that drastic compositional shifts in tongue microbiota occur before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years. These results may be helpful for preventing the development of various diseases associated with oral microbiota throughout life.

Preliminary Evaluation of Anti-Listerial Bacteriocin-like Peptide Produced by Enterococcus lactis PMD74 Isolated from Ezine Cheese

Enterococcus lactis PMD74 is a novel strain with a notably high antimicrobial activity. The present study evaluated the anti-listerial effect of a bacteriocin-like peptide (BLIP) produced by E. lactis PMD74 isolated from Ezine cheese (PDO). The strain was screened for its antimicrobial activity against 22 indicator strains using both agar spot and well diffusion methods. We observed that the neutralized cell-free supernatant (CFS) of E. lactis PMD74 exhibited varying levels of antimicrobial activity against both closely and distantly related pathogenic strains, with the highest activity displayed against Listeria monocytogenes strains. Although thermostable and resistant to lysozyme treatment, BLIP could be completely inactivated by trypsin, proteinase K, and α-chymotrypsin treatments. BLIP production starts in the early exponential growth phase of E. lactis PMD74 (3 h incubation, 400 AU mL–1) and reaches its maximal production (6400 AU mL–1) at the end of the exponential growth phase. Moreover, it is stable in the pH range of 2.0 to 7.0. The treatment of cultures of L. monocytogenes ATCC 7644 and Escherichia coli ATCC 26922 with sterilized CFS exhibited bactericidal and bacteriostatic effects, respectively. Furthermore, co-inoculation of L. monocytogenes ATCC 7644 and E. lactis PMD74 in skim milk led to complete loss of viability of L. monocytogenes ATCC 7644. These findings suggest that BLIP produced by E. lactis PMD74 could serve as a promising food preservative agent owing to its bactericidal and bacteriostatic properties.

Pseudomonas putida KT2440 metabolism undergoes sequential modifications during exponential growth in a complete medium as compounds are gradually consumed.

Summary Pseudomonas putida is a soil bacterium with a versatile and robust metabolism. When confronted with mixtures of carbon sources, it prioritizes the utilization of the preferred compounds, optimizing metabolism and growth. This response is particularly strong when growing in a complex medium such as LB. This work examines the changes occurring in P. putida KT2440 metabolic fluxes, while it grows exponentially in LB medium and sequentially consumes the compounds available. Integrating the uptake rates for each compound at three different moments during the exponential growth with the changes observed in the proteome, and with the metabolic fluxes predicted by the iJN1411 metabolic model for this strain, allowed the metabolic rearrangements that occurred to be determined. The results indicate that the bacterium changes significantly the configuration of its metabolism during the early, mid and late exponential phases of growth. Sugars served as an energy source during the early phase and later as energy and carbon source. The configuration of the tricarboxylic acids cycle varied during growth, providing no energy in the early phase, and turning to a reductive mode in the mid phase and to an oxidative mode later on. This work highlights the dynamism and flexibility of P. putida metabolism.

Exploring Evolutionary and Transmission Dynamics of HIV Epidemic in Serbia: Bridging Socio-Demographic With Phylogenetic Approach.

Previous molecular studies of Serbian HIV epidemic identified the dominance of subtype B and presence of clusters related HIV-1 transmission, in particular among men who have sex with men (MSM). In order to get a deeper understanding of the complexities of HIV sub-epidemics in Serbia, epidemic trends, temporal origin and phylodynamic characteristics in general population and subpopulations were analyzed by means of mathematical modeling, phylogenetic analysis and latent class analysis (LCA). Fitting of the logistic curve of trends for a cumulative annual number of new HIV cases in 1984–2016, in general population and MSM transmission group, was performed. Both datasets fitted the logistic growth model, showing the early exponential phase of the growth curve. According to the suggested model, in the year 2030, the number of newly diagnosed HIV cases in Serbia will continue to grow, in particular in the MSM transmission group. Further, a detailed phylogenetic analysis was performed on 385 sequences from the period 1997–2015. Identification of transmission clusters, estimation of population growth (Ne), of the effective reproductive number (Re) and time of the most recent common ancestor (tMRCA) were estimated employing Bayesian and maximum likelihood methods. A substantial proportion of 53% of subtype B sequences was found within transmission clusters/network. Phylodynamic analysis revealed Re over one during the whole period investigated, with the steepest slopes and a recent tMRCA for MSM transmission group subtype B clades, in line with a growing trend in the number of transmissions in years approaching the end of the study period. Contrary, heterosexual clades in both studied subtypes – B and C – showed modest growth and stagnation. LCA analysis identified five latent classes, with transmission clusters dominantly present in 2/5 classes, linked to MSM transmission living in the capital city and with the high prevalence of co-infection with HBV and/or other STIs.Presented findings imply that HIV epidemic in Serbia is still in the exponential growth phase, in particular, related to the MSM transmission, with estimated steep growth curve until 2030. The obtained results imply that an average new HIV patient in Serbia is a young man with concomitant sexually transmitted infection.

Isolation and Characterization of Multidrug Resistance Aeromonas salmonicida subsp. salmonicida and Its Infecting Novel Phage ASP-1 from Goldfish (Carassius auratus).

In this study, Aeromonas salmonicida subsp. salmonicida was isolated, identified by 16S RNA sequencing and its potential lytic phage (ASP-1) was isolated and characterized. The bacterium was positive for virulence genes (ascV, fla, ahyB, gcaT, lip, alt and act) and phenotypic parameters (haemolysis, slime production, lipase activity, DNase test, gelatinase activity and protease activity) were tested. The bacterium was resistant to 27%, intermediate resistant to 14% and susceptible to 59% of tested common antibiotics. Transmission electron microscopy analysis revealed that lytic ASP-1 belongs to the Myoviridae family. The isolated phage was more specific against A. salmonicida subsp. salmonicida (efficiency of plating index = 1), but also had infectivity to A. hydrophila lab strain 1. The bacteriolytic effect of ASP-1 was tested at early exponential phase culture of A. salmonicida subsp. salmonicida, and bacteria growth was apparently decreased with time and MOI dependent manner. One-step growth of ASP-1 showed approximately 30min of latent period, 16 PFU/infected cells of burst size and 40min of rise period. The adsorption rate was determined as 3.61 × 108PFUmL-1min-1 for 3min, and rate decreased with time. The ASP-1 genome size was estimated to be approximately 55-60kD. The phage was stable over wide-range of temperatures, pH and salinity, thus could withstand at severe environmental conditions, indicating that ASP-1 has a potential to develop as an alternative antibiotic touse in ornamental and aquaculture industry.

First Viruses Infecting the Marine Diatom Guinardia delicatula.

The marine diatom Guinardia delicatula is a cosmopolitan species that dominates seasonal blooms in the English Channel and the North Sea. Several eukaryotic parasites are known to induce the mortality of this species. Here, we report the isolation and characterization of the first viruses that infect G. delicatula. Viruses were isolated from the Western English Channel (SOMLIT-Astan station) during the late summer bloom decline of G. delicatula. A combination of laboratory approaches revealed that these lytic viruses (GdelRNAV) are small tailless particles of 35–38 nm in diameter that replicate in the host cytoplasm where both unordered particles and crystalline arrays are formed. GdelRNAV display a linear single-stranded RNA genome of ~9 kb, including two open reading frames encoding for replication and structural polyproteins. Phylogenetic relationships based on the RNA-dependent-RNA-polymerase gene marker showed that GdelRNAV are new members of the Bacillarnavirus, a monophyletic genus belonging to the order Picornavirales. GdelRNAV are specific to several strains of G. delicatula. They were rapidly and largely produced (<12 h, 9.34 × 104 virions per host cell). Our analysis points out the host's variable viral susceptibilities during the early exponential growth phase. Interestingly, we consistently failed to isolate viruses during spring and early summer while G. delicatula developed important blooms. While our study suggests that viruses do contribute to the decline of G. delicatula's late summer bloom, they may not be the primary mortality agents during the remaining blooms at SOMLIT-Astan. Future studies should focus on the relative contribution of the viral and eukaryotic pathogens to the control of Guinardia's blooms to understand the fate of these prominent organisms in marine systems.

Ocular Component Development during Infancy and Early Childhood.

The study fills an important gap by providing a longitudinal description of development of the major structural and optical components of the human eye from 3 months to nearly 7 years of age. Normative development data may provide insights into mechanisms for emmetropization and guidance on intraocular lens power calculation. The purpose of this study was to describe the pattern of development of refractive error and the ocular components from infancy through early childhood. Cycloplegic retinoscopy (cyclopentolate 1%), keratophakometry, and ultrasonography were performed longitudinally on between 162 and 293 normal birth weight infants at 0.25, 0.75, 1.5, 3, 4.5, and 6.5 years of age. Refractive error and most ocular components displayed an early exponential phase of rapid development during the first 1 to 2 years of life followed by a slower quadratic phase. Anterior and vitreous chamber depths, axial length, and crystalline lens radii increased at every visit. The crystalline lens thinned throughout the ages studied. The power of the cornea showed an early decrease, then stabilized, whereas the crystalline lens showed more robust decreases in power. The crystalline lens refractive index followed a polynomial growth and decay model, with an early increase followed by a decrease starting at 1 to 2 years of age. Refractive error became less hyperopic and then was relatively stable after 1 to 2 years of age. Axial lengths increased by 3.35 ± 0.64 mm between ages 0.25 and 6.5 years, showed uniform rates of growth across the range of initial values, and were correlated with initial axial lengths (r = 0.44, P < .001). Early ocular optical and structural development appears to be biphasic, with emmetropization occurring within the first 2 years of infancy during a rapid exponential phase. A more stable refractive error follows during a slower quadratic phase of growth when axial elongation is compensated primarily by changes in crystalline lens power.

Proposal of a new thraustochytrid genus Hondaea gen. nov. and comparison of its lipid dynamics with the closely related pseudo-cryptic genus Aurantiochytrium

Thraustochytrids are marine protists highly ecologically relevant in mangrove environments. The family Thraustochytriaceae underwent profound taxonomical rearrangements in the last decade, with the description and emendation of several genera. Here, we identified two new thraustochytrid strains (CCAP 4062/1 and CCAP 4062/3) collected from the same mangrove environment in Mayotte Island (Indian Ocean) and representative of two sister clades in the phylogenetic Aurantiochytrium super clade. Phylogenomic (on 2389 genes) and phylogenetic analyses on 18S rDNA sequences led us to propose the description of a new genus, Hondaea gen. nov. (CCAP 4062/3), closely related and pseudo-cryptic to Aurantiochytrium (CCAP 4062/1). Compared to Aurantiochytrium, Hondaea did not produce amoeboid cells and its zoospores were smaller. Chemotaxonomical traits, such as fatty acid, sterol, and carotenoid profiles measured along the growth curves, validated the new genus description. Genome sequencing and manual annotation of lipid metabolism genes revealed similar pathways in both strains. However, such pathways showed different dynamics during the growth phases. Aurantiochytrium accumulated carotenoids (canthaxanthin) and large amounts of triacylglycerols enriched in ω3-docosahexaenoic acid in the stationary phase, while squalene and free cholesterol increased during the early exponential phase. In contrast, Hondaea accumulated low amounts of triacylglycerols enriched in odd and saturated fatty acids during the early exponential phase, whereas free-sterol and carotenoid contents were little affected. These results suggest that these genera evolved independently, although phylogenetically and ecologically closely related. This comparative study also showed that the biotechnological potential of thraustochytrids cannot be deduced solely from phylogenetic and genomic analyses.

Characterization and pyrolysis behavior of the green microalga Micractinium conductrix grown in lab-scale tubular photobioreactor using Py-GC/MS and TGA/MS

Nowadays, microalgae are widely discussed as a promising feedstock for biofuel production. For higher crude bio-oil yield with good quality, microalgal biomass productivity and bio-oil characteristics are essential parameters. However, the same microalgal species has different chemical compositions at different growth phases. Therefore, the present study aimed to identify the best growth phase for high biomass productivity and optimal bio-oil production from the green microalga Micractinium conductrix via Py-GC/MS and TGA/MS analysis. M. conductrix was grown in a tubular photobioreactor and harvested at early exponential phase (EEP), middle exponential phase (MEP), late exponential phase (LEP) and stationary phase (STP). LEP showed the maximum significant (P ≤ 0.05) biomass productivity of 0.058 ± 0.004 g L−1 d-1, with maximum significant lipid and carbohydrate contents (28.7 ± 1.1 and 42.9 ± 1.2%dw, respectively). TGA/MS results confirmed that biomass harvested at MEP and LEP showed higher extent of conversion or mass loss reaction via thermal degradation with the lowest residual solid products. In addition, the hydrocarbon fragments in gaseous products (H2, C2H6, CH4, C2H4) from TGA/MS analysis were found to be released more abundantly at LEP. Moreover, Py-GC/MS results revealed that thermal decomposition of biomass harvested at LEP resulted in the highest significant relative contents of aliphatic hydrocarbons (41.2%) with lowest nitrogen-containing compounds (6.3%). The present study showed the significant impact of harvest time of microalgae on products characteristics of thermal decomposition and nominated LEP as the optimum growth phase to harvest M. conductrix for upgraded bio-oil production.

Optimization of electroporation-based multiple pulses and further improvement of transformation efficiency using bacterial conditioned medium for Nannochloropsis salina

Development of transgenic strains of microalgae is necessary to obtain microalgal strains that are capable of producing high levels of oils and a variety of bio-products. However, microalgae have a much lower transformation efficiency in comparison to other micro-organisms due to the presence of thick cell walls. Here, a two-pronged approach for optimizing the electroporation parameters combined with the application of secondary metabolites from myxobacteria to weaken the cell wall was used to maximize the transformation efficiency of Nannochloropsis salina (Eustigmatophyceae). When the electroporation parameters were set at 50 pulses of square waves at a field strength of 12,000 V cm−1, a transformation efficiency of up to 53 per 108 cells was achieved for N. salina in the early exponential phase of growth. Next, cultivation of the microalgae using bacterial conditioned F2N medium containing 10% supernatant of the myxobacteria strain DZ2 resulted in a moderate inhibition of the growth rate and distinctive morphological changes. Flow cytometry confirmed an increase in the overall cell size as well as granularity. When the bacterial medium conditioned cells were employed for transformation under optimized electroporation conditions, the transformation efficiency improved 2.71-fold up to 144 per 108 cells. Taken together, a combinatory approach for electroporation using high field strength square wave pulses and conditioning cells with myxobacterial supernatant is a promising method for the efficient genetic transformation of microalgae.

Variations in morphology, growth, and toxicity among strains of the Prorocentrum lima species complex isolated from Cuba and Brazil

Benthic dinoflagellates belonging to the genus Prorocentrum are known to produce diarrhetic toxins such as okadaic acid (OA) and its analogues, dinophysistoxins (DTXs), as well as prorocentrolides and other unidentified fast-acting toxins. The present study is a comparative analysis focused on the morphology, genetic, growth, toxin production, and toxicity by strains belonging to the Prorocentrum lima species complex, isolated from different regions along the western Atlantic coast. While cell dimensions (38–45 × 24–30 μm) and shape (ovoid) were similar between strains from Cuba and Recife (Northeast Brazil), cells of the strain from the estuarine complex of Paranagua Bay (South Brazil) were shorter (36–41 × 25–28 μm) and oblong to ovate-oblong (elliptical) in shape. This latter strain exhibited similar LSU rDNA and identical ITS1-5.8S rDNA-ITS2 sequences to those of P. cf. lima “morphotype 5,” which is closely related to Prorocentrum caipirignum by LSU, but separated from it by ITS. This southern Brazilian strain attained the highest growth rate (0.34 ± 0.01 div day−1) and cell densities (11.2 × 105 cell mL−1) in batch culture. Intracellular OA concentrations were higher for the other two strains during the late exponential and stationary phase, but similar for all strains (9.50–10.06 pg cell−1) at the early exponential growth phase; consistently lower levels of DTX-1 were produced by two strains, except the one from Recife. Finally, live cells of the strains from Cuba and Recife were more toxic to A. salina metanauplii, whereas the southern Brazilian strain exhibited higher culture medium toxicity. The contrasts in growth and toxicity potential revealed for these morphologically and genetically distinct Prorocentrum strains might be relevant for the local management of diarrhetic poisoning outbreaks in shellfish harvesting and aquaculture sites, including in estuarine areas.

UV-C suppression on hazardous metabolites in Microcystis aeruginosa: Unsynchronized production of microcystins and odorous compounds at population and single-cell level

The effectiveness of UV-C towards the toxin and odor of M. aeruginosa at population and single cell levels were investigated in three ways. In the absence of UV-C, MC-LR and β-cyclocitral production show similar pattern of incremental rate with growth rate on population level shown as intracellular concentrations of MC-LR (CMC) and β-cyclocitral (CBCC), but the cellular quota of MC-LR (QMC) and β-cyclocitral (QBCC) at single-cell level reached the maximum values, 37.5 ± 1.2 fg cell−1 and 4.3 ± 0.1 fg cell−1, just after the early exponential phase. Second, upon UV-C irradiation, the CMC consistently decreased by 10–41% with increase of UV-C dosage (50–200 mJ cm-2) while CBCC increased by 2–14%. Third, during the 14 days’ post-UV incubation, UV-C at 75–200 mJ cm-2 induced remarkable suppressing effects on both CMC and CBCC for 3–14 days. The suppressing effects on QMC and QBCC were induced by UV-C at 100 mJ cm-2 and above, with shorter suppressing periods by 1–4 days and lower decremental rates by 21%–30% than that of CMC and CBCC, indicating interruptions on biosynthesis processes partially contribute to suppression effects of CMC and CBCC. The suppression effect on either CBCC or QBCC, with higher decrement rates while lower recovery rates, was more severe than CMC and QMC.

Construction and co-cultivation of two mutant strains harboring key precursor genes to produce prodigiosin

The biosynthesis of prodigiosin (PG) from Serratia marcescens involves the coupling of a bipyrrole, 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC), with a monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP), and formation of a linear tripyrrole (PG). We constructed mutant strains in which either the MBC biosynthesis by S.marcescens BMJ816 or the MAP biosynthesis by S.marcescens AMJ817. S.marcescens BMJ816 and AMJ817 confirmed that they lose the ability to synthesize PG when they are cultivated alone. An experiment was also conducted in which cultures of the two mutant strains were grown to the early exponential phase in a semi-defined medium, and one suspension culture was inoculated with the other. This approach yielded 103mg/L PG. The findings suggest that the addition of precursors may enhance PG production by microorganisms.