Thermal acclimation in clownfish: An integrated biomarker response and multi-tissue experimental approach

The effects of increased temperature were tested in Amphiprion ocellaris, using a cellular diagnostics approach (in several tissues) combined with an organismal approach (body condition). Clownfish were exposed to a one month experiment following two temperature treatments: control (26 °C) and elevated temperature (30 °C). Fish were sampled at 0, 7, 14, 21 and 28 days for (1) assessment of stress biomarkers (catalase, lipid peroxidation, glutathione-S-transferase, superoxide dismutase, acetylcholinesterase, heat shock protein 70 kDa and ubiquitin – in brain, gills, liver, intestine and muscle), (2) estimation of integrated biomarker response index based on the biomarkers tested and (3) assessment of Fulton’s K index. Results show all biomarkers except acetylcholinesterase responded consistently and significantly to elevated temperature across tissue types suggesting they are suitable indicators of thermal stress in A. ocellaris. Biomarker levels were tissue-specific, and in addition, the most reactive tissues to temperature were muscle, gills and liver which suggest that highly oxygenated tissues seem to be the most responsive under thermal stress. The most responsive sampling times to increased temperature were T7 and T28: thermal stress was observed after 7 days of exposure (biomarker levels increased), then a pattern of decrease in biomarker levels towards the end of the experiment was observed, which may suggest fish were able to acclimate to exposure conditions. This indicates that A. ocellaris probably lives far from its upper thermal limit and is capable of adjusting the protein quality control system and enzymes’ activities to protect cell functions under elevated temperatures. The temperature treatment did not significantly influence body condition of the animals but biomarkers were negatively correlated to wet body weight. This suggests that thermal acclimation incurs at some energetic cost. In conclusion, these results suggest that this coral reef fish species presents a significant acclimation potential under ocean warming scenarios of +4 °C.     

Mitochondrial distribution patterns in canine oocytes as related to the reproductive cycle stage

This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n = 2), follicular phase (F, n = 4), ovulation (O, n = 2), early luteal (EL, n = 7) and mid/late luteal phase (MLL, n = 2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110 h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (I) small aggregates diffused throughout the cytoplasm; (II) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II + III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P < 0.05; O vs. A: P < 0.001), in EL (61%; O vs. EL: P < 0.01), or in MLL (0%; F vs. MLL: P < 0.05; O vs. MLL: P < 0.001). Fluorescence intensity did not vary according to mt distribution pattern except that it was lower in oocytes recovered in EL phase and showing small mt aggregations (P < 0.001). The majority of ovulated MII stage oocytes (79%) showed diffused tubular mt network. We conclude that mt distribution pattern of canine ovarian immature oocytes changes in relation to reproductive cycle stage and that patterns observed in oocytes recovered from bitches in periovulatory phases are heterogeneous and similar to those of in vivo matured oocytes.     

Multiple sclerosis: Identification and clinical evaluation of novel CSF biomarkers

Multiple sclerosis (MS) is a neuro-inflammatory and neurodegenerative disease that results in damage to myelin sheaths and axons in the central nervous system and which preferentially affects young adults. We performed a proteomics-based biomarker discovery study in which cerebrospinal fluid (CSF) from MS and control individuals was analyzed (n = 112). Ten candidate biomarkers were selected for evaluation by quantitative immunoassay using an independent cohort of MS and control subjects (n = 209). In relapsing–remitting MS (RRMS) patients there were significant increases in the CSF levels of alpha-1 antichymotrypsin (A1AC), alpha-1 macroglobulin (A2MG) and fibulin 1 as compared to control subjects. In secondary progressive MS (SPMS) four additional proteins (contactin 1, fetuin A, vitamin D binding protein and angiotensinogen (ANGT)) were increased as compared to control subjects. In particular, ANGT was increased 3-fold in SPMS, indicating a potential as biomarker of disease progression in MS. In PPMS, A1AC and A2MG exhibit significantly higher CSF levels than controls, with a trend of increase for ANGT. Classification models based on the biomarker panel could identify 70% of the RRMS and 80% of the SPMS patients correctly. Further evaluation was conducted in a pilot study of CSF from RRMS patients (n = 36), before and after treatment with natalizumab.     

Effect of thermal stress on metabolic and oxidative stress biomarkers of Hoplosternum littorale (Teleostei,Callichthyidae)

The present study aimed to investigate in Hoplosternum littorale (Hancock, 1828) the effects of different water temperatures (10 °C, 25 °C-control group- and 33 °C) on physiologic and metabolic traits following acute (1 day) and chronic (21 days) exposures. We analyzed several biomarker responses in order to achieve a comprehensive survey of fish physiology and metabolism under the effect of this natural stressor. We measured morphological indices, biochemical and hematological parameters as well as oxidative stress markers. To evaluate energy consumption, muscle and hepatic total lipid, protein and glycogen concentrations were also quantified. Extreme temperatures exposures clearly resulted in metabolic adjustments, being liver energy reserves and plasma metabolites the most sensitive parameters detecting those changes. We observed reduced hepatosomatic index after acute and chronic exposure to 33 °C while glycogen levels decreased at both temperatures and time of exposure tested. Additionally, acute and chronic exposures to 10 °C increased liver lipid content and plasma triglycerides. Total protein concentration was higher in liver and lower in plasma after chronic exposures to 10 °C and 33 °C. Acute exposition at both temperatures caused significant changes in antioxidant enzymes tested in the different tissues without oxidative damage to lipids. Antioxidant defenses in fish failed to protect them when they were exposed for 21 days to 10 °C, promoting higher lipid peroxidation in liver, kidney and gills. According to multivariate analysis, oxidative stress and metabolic biomarkers clearly differentiated fish exposed chronically to 10 °C. Taken together, these results demonstrated that cold exposure was more stressful for H. littorale than heat stress. However, this species could cope with variations in temperature, allowing physiological processes and biochemical reactions to proceed efficiently at different temperatures and times of exposure. Our study showed the ability of H. littorale to resist a wide range of environmental temperatures and contributes for the understanding of how this species is adapted to environments with highly variable physicochemical conditions.     

Neutrophil CD64 expression and serum IL-8: Sensitive early markers of severity and outcome in sepsis

The aim of the present study was to investigate which biomarker/s reliably assess severity and mortality early in the sepsis process. In 47 critically-ill patients within the 24 h of septic onset, Interleukins (IL)-8, -1β, -6, -10, and -12p70, tumor necrosis factor-α (TNF-α), procalcitonin (PCT) and C-reactive protein (CRP) were measured in serum. Additionally, CD64 expression was measured in neutrophils. In early sepsis, neutrophil CD64 expression and IL-8 levels are the only biomarkers that increased with sepsis severity, differentiating disease stages: sepsis, severe sepsis and septic shock (p < 0.001). The biomarkers that best evaluate the severity of sepsis (via APACHE II) were CD64, IL-8 and IL-6 (p < 0.01), and the severity of organ failure (via SOFA) were CD64 and IL-8 (p < 0.01). CD64 expression and IL-8 levels were associated with mortality within 28-days (OR = 1.3, p = 0.01 for CD64 and OR = 1.26, p = 0.024 for IL-8 by logistic regression analysis) and ROC curve analysis showed high sensitivity and specificity for predicting sepsis stages and the 28 day mortality. We conclude that there is an early increase of neutrophil CD64 expression and IL-8 levels during sepsis. Based on this single measurement it is possible to reliably assess the stage, detect the severity and predict the 28-day mortality of sepsis.     

Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout,Oncorhynchus mykiss

The effect of long-term (30 days) exposure to PCZ (0.2, 50, and 500 μg l^?1) on intestine-related biochemical markers in rainbow trout was investigated. Multiple biomarkers were measured, including digestive enzymes (proteolytic enzymes and amylase), antioxidant responses (TBARS, CP, SOD, CAT, GR and GPx) and energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase). Exposure to 500 μg l^?1 PCZ led to significantly inhibited (p < 0.01) proteolytic enzyme and amylase activity. Activities of the antioxidant enzymes SOD, CAT, and GPx gradually increased at lower PCZ concentrations (0.2 and 50 μg l^?1). At the highest concentration (500 μg l^?1), oxidative stress was apparent as significant higher (p < 0.05) lipid peroxidation and protein carbonyls, associated with an inhibition of antioxidant enzymes activity. Moreover, energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase) were significantly inhibited (p < 0.01) in the intestines of fish exposed to 500 μg l^?1 PCZ, compared with controls. We suggest that long-term exposure to PCZ could result in several responses in intestine-related biochemical markers, which potentially could be used as indicators for monitoring residual PCZ present in the aquatic environment.     

Biological and molecular responses of Chironomus riparius (Diptera,Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10 μg L^? 1 of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10 μg L^? 1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1 μg L^? 1 of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24 h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10 μg L^? 1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.     

A let-7 microRNA polymorphism in the KRAS 3′-UTR is prognostic in oropharyngeal cancer

IntroductionThis study aimed to investigate the effect of genetic polymorphisms in miRNA sequences, miRNA target genes and miRNA processing genes as additional biomarkers to HPV for prognosis in oropharyngeal squamous cell carcinoma (OPSCC) patients. Secondarily, the prevalence of HPV-associated OPSCC in a European cohort was mapped.MethodsOPSCC patients (n = 122) were genotyped for ten genetic polymorphisms in pre-miRNAs (pre-mir-146a, pre-mir-196a2), in miRNA biosynthesis genes (Drosha, XPO5) and in miRNA target genes (KRAS, SMC1B). HPV status was assessed by p16 immunohistochemistry (IHC) and high-risk HPV in situ hybridization (ISH) or by p16 IHC and PCR followed by enzyme-immunoassay (EIA). Overall and disease specific survival were analysed using Kaplan–Meier plots (log-rank test). Cox proportional hazard model was used to calculate hazard ratios (HR).ResultsThe overall HPV prevalence rate in our Belgian/Dutch cohort was 27.9%. Patients with HPV⁺ tumours had a better 5-years overall survival (78% vs. 46%, p = 0.001) and a better 5-years disease specific survival (90% vs. 70%, p = 0.016) compared to patients with HPV⁻ tumours. In multivariate Cox analysis including clinical, treatment and genetic parameters, HPV negativity (HR = 3.89, p = 0.005), advanced T-stage (HR = 1.81, p = 0.050), advanced N-stage (HR = 5.86, p = 0.001) and >10 pack-years of smoking (HR = 3.45, p = 0.012) were significantly associated with reduced overall survival. The variant G-allele of the KRAS-LCS6 polymorphism was significantly associated with a better overall survival (HR = 0.40, p = 0.031).ConclusionsOur results demonstrate that OPSCC patients with the KRAS-LCS6 variant have a better outcome and suggest that this variant may be used as a prognostic biomarker for OPSCC.     

Differential Expression of Circular RNAs in Glioblastoma Multiforme and Its Correlation with Prognosis

OBJECTIVE: The present study aimed to explore the expression profiles of circular RNAs (circRNAs) in glioblastoma multiforme (GBM) in an attempt to identify potential core genes in the pathogenesis of this tumor. METHODS: Differentially expressed circRNAs were screened between tumor tissues from five GBM patients and five normal brain samples using Illumina Hiseq. Bioinformatics analysis was used to analyze their potential function. CircBRAF was further detected in different WHO grades glioma tissues and normal brain tissues. Kaplan-Meier curves and multivariate Cox's analysis were used to analyze the association between circBRAF expression level and prognosis of glioma patients. RESULTS: A total of 1411 differentially expressed circRNAs were identified in GBM patients including 206 upregulated circRNAs and 1205 downregulated circRNAs. Differential expression of circRNAs was closely associated with the biological process and molecular function. The downregulated circRNAs were mainly associated with ErbB and Neurotrophin signaling pathways. Moreover, the expression level of circBRAF in normal brain tissues was significantly higher than that in glioma tissues (P < .001). CircBRAF was significantly lower in glioma patients with high pathological grade (WHO III & IV) than those with low grade (WHO I & II) (P < .001). Cox analysis revealed that high circBRAF expression was an independent biomarker for predicting good progression-free survival and overall survival in glioma patients (HR = 0.413, 95% CI 0.201-0.849; HR = 0.299, 95% CI 0.135-0.661; respectively). CONCLUSION: The present study identified a profile of dysregulated circRNAs in GBM. Bioinformatics analysis showed that dysregulated circRNAs might be associated with tumorigenesis and development of GBM. In addition, circBRAF could severe as a biomarker for predicting pathological grade and prognosis in glioma patients.     

Growth and antrum formation of bovine primary follicles in long-term culture in vitro

Successful antral formation in vitro from bovine preantral follicles (145–170 μm) has been described previously, but antrum formation from the primary follicle (50–70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50–70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E₂), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E₂ + bFGF or FSH + LH + E₂ + bFGF + EGF (p < 0.05). An addition of 50 ng/ml bFGF or bFGF + 25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF + 25 ng/ml EGF to media containing FSH + LH + E₂ turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.     

Homologous expression,purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416

The lipase secreted by Burkholderia cepacia ATCC 25416 was particularly attractive in detergent and leather industry due to its specific characteristics of high alkaline and thermal stability. The lipase gene (lipA), lipase chaperone gene (lipB), and native promoter upstream of lipA were cloned. The lipA was composed of 1095 bp, corresponding to 364 amino acid residues. The lipB located immediately downstream of lipA was composed of 1035 bp, corresponding to 344 amino acid residues. The lipase operon was inserted into broad host vector pBBRMCS1 and electroporated into original strain. The homologous expression of recombinant strain showed a significant increase in the lipase activity. LipA was purified by three-step procedure of ammonium sulfate precipitation, phenyl-sepharose FF and DEAE-sepharose FF. SDS-PAGE showed the molecular mass of the lipase was 33 kDa. The enzyme optimal temperature and pH were 60 °C and 11.0, respectively. The enzyme was stable at 30–70 °C. After incubated in 70 °C for 1 h, enzyme remained 72% of its maximal activity. The enzyme exhibited a good stability at pH 9.0–11.5. The lipase preferentially hydrolyzed medium-chain fatty acid esters. The enzyme was strongly activated by Mg²⁺, Ca²⁺, Cu²⁺, Zn²⁺, Co²⁺, and apparently inhibited by PMSF, EDTA and also DTT with SDS. The enzyme was compatible with various ionic and non-ionic surfactants as well as oxidant H₂O₂. The enzyme had good stability in the low- and non-polar solvents.     

Tensiomyography of selected lower-limb muscles in professional soccer players

Tensiomyography is a non-invasive method of neuromuscular assessment used to measure muscle action characteristics, muscle tone, and muscle fiber type, and provides information on acute and chronic responses of muscle to different training loads. The aims of the present study were: to analyse differences in muscle response and mechanical characteristics of two major muscles of the lower extremity in a large group of Spanish soccer players according to playing position, and to provide group norms against which clinical findings may be compared. Data were collected from 78 professional soccer players (age 26.6 ± 4.4 years; height: 179.2 ± 5.3 cm; body mass: 75.8 ± 5.3 kg). Tensiomyography was recorded from the rectus femoris (RF) and biceps femoris (BF) muscles after 2 days without take part in any strenuous exercise or training. Five tensiomyographic parameters were analyzed: maximal displacement (D_m), contraction time (T_c), sustain time (T_s), delay time (T_d), and half-relaxation time (T_r). A good to excellent intra-session reliability was found for all contractile parameters (ICC ranged from 0.78 to 0.95). No significant differences between players of any position were observed in absolute values of BF. However, significant differences were observed for T_c, T_r and T_s between the different playing positions on RF (P < 0.05, effect size ranged from 1.3 to 1.6). Professional soccer players showed muscles with ability to rapidly generate force during contractions. The neuromuscular profile provided could help in identifying the normative data that are important for the different positions in order to optimize the training and recovery process of each individual player.     

Predominance of terrestrial organic matter in sediments from a cyanobacteria- blooming hypereutrophic lake

Although an understanding of the quantity and quality of sedimentary organic matter (SOM) pools is necessary to design sound environmental management strategies for lacustrine systems, the characterization of organic matter sources and the assessment of their relative contributions to eutrophic and inland lake sediments remain insufficient. In this study, the contribution of potential organic matter sources to sediments in shallow and hypereutrophic lake Taihu, China was assessed on the molecular level using source-specific fatty acid biomarkers. The results indicated that SOM was composed mainly of terrestrial plants with a maximal contribution of 45.3 ± 2.4% to the total organic carbon, which accounted for approximately 66% among the determined organic matter sources. Evidence suggests the terrestrial plants remained in a fresh state in surface sediments: the correlation (R² = 0.62, p < 0.05) between bacterial and terrestrial plant carbon was strong. On the other hand, aquatic plant and bacterial carbon contributed 5–15% to the total organic carbon, which was followed by the faint contribution (<5% of total organic carbon) of algae-derived organic carbon including cyanobacteria, diatoms, and dinoflagellates. The results provided details of the contributions of SOM sources, illustrating the usefulness of fatty acid biomarkers in discriminating organic matter sources within lake environments. Although organic matter sources of sediments varied in spatial and temporal patterns, the strong correlation between terrestrial plant and total organic carbon (R² = 0.60, p < 0.05) indicates that terrestrial plants were the dominant source in lake sediments.     

Bioaccumulation and biochemical response in South American native species exposed to zinc: Boosted regression trees as novel tool for biomarkers selection

The aim of this study was to evaluate the response of a wide battery of biomarkers in two native species, the freshwater shrimp Palaemonetes argentinus and the macrophyte Potamogeton pusillus, experimentally exposed to zinc in order to establish the potential use of selected species as bioindicators of aquatic pollution. For this purpose, we propose the use of integrated biomarker index (IBR) with a previous selection of biomarkers using boosted regression trees (BRTs) as a new tool in ecotoxicology. Organisms were collected from a reference site, acclimated and exposed at relevant environmental zinc levels (control, 5, 50 and 500 μg Zn L⁻¹) for 96 h. Biomarkers were measured in cephalothorax and abdomen of shrimp as well as in leaf, stem and root of plants.Significant zinc accumulation was observed in cephalothorax of P. argentinus from 50 μg Zn L⁻¹ and from 5 μg Zn L⁻¹ in stem and root of P. pusillus, when compared with control condition. Those effect biomarkers with significant differences among treatments were pre-selected to run out the BRTs model for each species. In P. argentinus, microsomal acetylcholinesterase activity, metallothioneins and superoxide dismutase activity measured in cephalothorax, as well as glutathione reductase activity in abdomen, showed the higher capacity to explain or predict the zinc exposure concentration. On the contrary, in P. pusillus, only chlorophyll a measured in leaf and H₂O₂ measured in root were the more representative of exposure concentrations, at least, within the biomarkers tested in the present study. Thereafter, IBR was calculated with the selected biomarkers in P. argentinus and showed in a sole value the organism stress, which also correlates with zinc exposure and accumulation.Natives species tested displayed a sensitive response to metal exposure, which represents an important characteristic for biomonitoring programs. Our findings suggest that the BRTs and IBR are useful and robust run tools to select the better biomarkers in toxicological studies and indicate the organism stress.     

Oxidative stress and DNA damage in relation to transition metals overload in Abu-Qir Bay,Egypt

The aim of the present study is to evaluate the transition metals overload in Abu-Qir Bay in Egypt, as compared to a less polluted area (reference area) through some biomarkers of oxidative stress. Catalase enzyme activity, malondialdehyde (MDA) concentration and DNA damage (number of apurinic/apyrimidinic sites) were the tested biomarkers. The levels of iron and copper in Mugil cephalus liver tissues were significantly higher in samples from the polluted area as compared to the reference area: Fe: 407 ± 38 vs. 216 ± 21 μg/g wet wt; p = 0.008, Cu: 54 ± 6 vs. 17.7 ± 4 μg/g wet wt; p = 0.0001. This could account for the observed increase in MDA concentration (15.7 ± 5.7 vs. 2.5 ± 0.5 U/g; p = 0.035), and the elevated number of AP sites (13.9 ± 2.6 vs. 0.37 ± 0.2 AP site/1 × 10⁵ bp; p = 0.0001). Similarly, the activity of catalase enzyme responsible for the cellular defense was significantly high (58.3 ± 12.2 vs. 28.4 ± 4.0 U/mg; p = 0.032). The present data indicated a clear relationship between the pollution degree of the above marine environment and both biochemical and molecular responses of the piscine system.     

Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was autotrophically cultivated for biomass production in repeated fed-batch process using urea as nitrogen source, with the aim of making large-scale production easier, increasing cell productivity and then reducing the production costs. It was investigated the influence of the ratio of renewed volume to total volume (R), the urea feeding time (t_f) and the number of successive repeated fed-batch cycles on the maximum cell concentration (X_m), cell productivity (P_x), nitrogen-to-cell conversion yield (Y_x/n), maximum specific growth rate (μ_m) and protein content of dry biomass. The experimental results demonstrated that R = 0.80 and t_f = 6 d were the best cultivation conditions, being able to simultaneously ensure, throughout the three fed-batch cycles, the highest average values of three of the five responses (X_m = 2101 ± 113 mg L^?1, P_x = 219 ± 13 mg L^?1 d^?1 and Y_x/n = 10.3 ± 0.8 g g^?1).