Multi-stage glucose/pachymaran co-feeding enhanced endo-ß-1,3-glucanase production by Trichoderma harzianum via simultaneous increases in cell concentration and inductive effect.

Endo-ß-1,3-glucanase is used to hydrolyze curdlan in a wide range of oligosaccharides production processes. Using pachymaran as the sole carbon source resulted in an endo-ß-1,3-glucanase activity of 86.1 U/mL and an Eendo/Etotal ratio of 0.43, which were 3.2 and 1.65 folds of the values from control (glucose as the sole carbon source), due to the inductive effect of pachymaran as a polysaccharide. However, the cell concentration decreased from 25 to 12 g/L during the late fermentation phase. Therefore, a novel multi-stage feeding strategy was developed wherein glucose was fed twice during the cell logarithmic growth phase (24 and 48 h) and pachymaran once during the early stage of the enzyme accumulation phase (72 h). Consequently, the cell concentration remained around 30 g/L during the late fermentation phase. Endo-ß-1,3-glucanase activity and Eendo/Etotal reached 160.0 U/mL and 0.76, respectively, which were 6.0 and 2.92 folds of the values from control. In addition, three typical polysaccharides with ß-1,3-linked glucose residues were successfully hydrolyzed by endo-ß-1,3-glucanase to produce multifunctional ß-1,3-oligoglucosides.

Towards Understanding the Mechanism of Heavy Metals Immobilization in Biochar Derived from Co-pyrolysis of Sawdust and Sewage Sludge.

Biochar was prepared by mixing sewage sludge with sawdust via a co-pyrolysis with different mixture ratios and temperatures. The results showed that the sawdust addition resulted in a lower yield of biochar with higher C content. The total concentrations of Pb and Cd in biochar were reduced. Besides, pyrolysis can transform the potentially toxic Pb and Cd to stable fractions. However the sawdust addition had slight influence on the chemical forms of Pb and Cd in the biochar. The biochar with 50% sawdust at 600°C exhibited a remarkable reduction of the leachable metal concentrations. The possible transformation mechanisms of Pb and Cd were inferred as the formation of aluminum and silicon-containing minerals. These results provide insights into the influence of sawdust addition on the characteristics of biochar and the possible Pb and Cd immobilization mechanisms during co-pyrolysis process.

A mouse model of a novel missense mutation (Gly83Arg) in a Chinese kindred manifesting vitreous amyloidosis only.

The purpose of this study is to establish a mouse model of transthyretin (TTR) Gly83Arg gene mutation by the technique of gene targeting for research on hereditary vitreous amyloidosis (HVA) and to confirm whether this point mutation is a genetic feature of HVA. A vector (pBR322-MK-TTR) was constructed to target ES cells. The successfully transfected ES cells were used for blastocyst injection, thus generating F0. F0 and Flp mice were mated to generate F1 (TTR+/-, Flp +/-) mice that lacked the neo gene but carried the Flp gene. F1 mice were mated with C57BL/6N wild type mice to generate F2 (TTR+/-) mice. F3 homozygous and heterozygous mice were generated by mating F2 mice with each other. PCR and sequencing were performed for F3 mice. Amyloid was detected using Congo red stain and polarized light. Immunohistochemistry was used to detect the expression of TTR in the tissues. Quantitative fluorescent PCR and Western blotting were used to detect the expression of TTR mRNA and TTR protein, respectively. Two F0-generation, 2 F1-generation and 15 F3-generation mice were obtained. The gene sequencing of F3 mice showed TTR Gly83Arg mutation. When examined with Congo red and polarized light, the vitreous of TTR Gly83Arg mutant mice tested positive for amyloid. The hearts, livers, brains and kidneys of the experimental group and control group were all negative by Congo red staining. Immunohistochemical staining showed that the vitreous of TTR Gly83Arg mutant mice and the livers of the control mice were positive, but the kidneys, hearts and brains of both groups were negative. Quantitative fluorescent PCR showed that the mRNA expression of mutant mice was significantly lower than that of wild-type mice (F = 0.295, P = 0.023). Western blotting showed that the expression of TTR protein was significantly lower in the model mice than in the wild-type mice (t = 3.224, P = 0.018). TTR gene mutation is indeed a molecular characteristic of HVA and manifest in the eye disease only. A C57BL/6 mouse line carrying the TTR Gly83Arg gene mutation was successfully established. This strain of mice can be used for the study of HVA.

Keyhole epilepsy surgery: corticoamygdalohippocampectomy for mesial temporal sclerosis.

Surgical approaches for medically refractory mesial temporal lobe epilepsy (MTLE) that previously have been reported include anterior temporal lobectomy (ATL), transcortical selective amygdalohippocampectomy, transsylvian amygdalohippocampectomy, and subtemporal amygdalohippocampectomy. Each approach has its advantages and potential pitfalls. The purpose of this report is to describe our technique of keyhole corticoamygdalohippocampectomy for patients with MTLE due to hippocampal sclerosis. Operations were performed through a 6-cm vertical linear incision and a low 2.5-cm keyhole craniotomy at the anterior squamous temporal bone. Resection of the anterior-most portions of the middle and inferior temporal gyri provided a cylinder-like corridor to the mesial temporal lobe. Identification of the temporal horn through a basal approach was followed by resection of the amygdala, uncus, and hippocampus-parahippocampal gyrus. This 9-year series included 683 patients with a minimum follow-up duration of 2 years. Surgery times were short (range, 1 h 35 min to 2 h 30 min). Only a small percentage of patients had complications (1.76%), and the rate of Engel Class I seizure-free outcome was 87%. No overt speech problems or visual field deficits were identified. Compared with the most popular conventional trans-middle temporal gyrus approach, this technique can make the operation easier, safer, and less traumatic to functional lateral neocortex.

Improving arachidonic acid fermentation by Mortierella alpina through multistage temperature and aeration rate control in bioreactor.

Effective production of arachidonic acid (ARA) using Mortierella alpina was conducted in a 30-L airlift bioreactor. Varying the aeration rate and temperature significantly influenced cell morphology, cell growth, and ARA production, while the optimal aeration rate and temperature for cell growth and product formation were quite different. As a result, a two-stage aeration rate control strategy was constructed based on monitoring of cell morphology and ARA production under various aeration rate control levels (0.6-1.8 vvm). Using this strategy, ARA yield reached 4.7 g/L, an increase of 38.2% compared with the control (constant aeration rate control at 1.0 vvm). Dynamic temperature-control strategy was implemented based on the fermentation performance at various temperatures (13-28°C), with ARA level in total cellular lipid increased by 37.1% comparing to a constant-temperature control (25°C). On that basis, the combinatorial fermentation strategy of two-stage aeration rate control and dynamic temperature control was applied and ARA production achieved the highest level of 5.8 g/L.

Intractable occipital lobe epilepsy: clinical characteristics, surgical treatment, and a systematic review of the literature.

PURPOSE: We reported our experience in the surgical treatment of a relatively large cohort of patients with occipital lobe epilepsy (OLE). We also carried out a systematic review of the literature on OLE. METHODS: Thirty-five consecutive patients who underwent occipital resection for epilepsy were included. Diagnoses were made following presurgical evaluations, including magnetic resonance imaging (MRI), fluorodeoxyglucose-positron emission tomography (FDG-PET), scalp video-electroencephalogram (EEG) monitoring, and intracranial EEG monitoring. At last follow-up, seizure outcome was classified using the Engel classification scheme. RESULTS: Twenty-five of 35 patients experienced/had experienced ≥1 type of aura before the seizure. Invasive recordings were used to define the epileptogenic area in 30 of 35 patients (85.7 %). All patients underwent occipital lesionectomies or topectomies. Histopathology revealed: cortical dysplasias, gliosis, dysembryoplastic neuroepithelial tumor, ganglioglioma, and tuberous sclerosis. After a mean follow-up of 44 months, 25 patients (71.4 %) were seizure free (Engel class I), 3 (8.6 %) rarely had seizures (Engel class II), 5 (14.3 %) improved more than 75 % (Engel class III), and 2 (5.7 %) had no significant improvement (Engel class IV). Preoperatively, 12 of 33 patients (36.4 %) had visual field deficits. Postoperatively, 25 patients (75.8 %) had new or aggravated visual field deficits. CONCLUSIONS: The management of OLE has been aided greatly by the availability of high-resolution diagnosis. Postoperative visual field deficits occur in a significant proportion of patients. Comprehensive intracranial EEG coverage of all occipital surfaces helps to define the epileptogenic area and preserve visual function, especially in cases of focal cortical dysplasia undetectable by MRI.

Design of highly sensitive phosphorescence sensor for determination of procaterol hydrochloride based on inhibition of KClO3 oxidation fluorescein isothiocyanate.

Procaterol hydrochloride (Prh) can inhibit KClO3 oxidation of fluorescein isothiocyanate (FITC) to form a non-phosphorescent compound, which causes room temperature phosphorescence (RTP) of FITC in the system to enhance sharply the linear relationship between ∆Ip and the Prh content. Thus, a rapid response and highly sensitive phosphorescence sensor for the determination of Prh has been developed based on the inhibiting effect of Prh on KClO3 oxidation of FITC. This simple, high sensitivity (detection limit (LD) calculated by 3Sb /k was 0.019 fg/spot, sample volume 0.40 µl, corresponding concentration 4.8 × 10(-14) g ml(-1) ) and selective sensor with a wide linear range (0.080-11.20 g/spot) has been applied to detect Prh in blood samples, and the results were consistent with those obtained by high-performance liquid chromatography (HPLC). Simultaneously, the mechanism of the phosphorescence sensor for the detection of Prh was also investigated using infrared spectroscopy.

A highly sensitive solid substrate room temperature phosphorimetry for carbaryl detection based on its activating effect on NaIO4 oxidizing fluorescein.

Fluorescein (HFin) could emit strong and stable room temperature phosphorescence (RTP) signal on polyamide membrane (PAM) using Pb(2+) as the ion perturber. Carbaryl could activate effect on NaIO4 oxidating HFin, which caused the RTP signal of the system to quench sharply. The phosphorescence intensity (ΔI p) of activating system higher 3.3 times (119.4/36.0) than that of non-activating system, and is directly proportional to the content of carbaryl. Thus, an activating solid substrate room temperature phosphorimetry (SSRTP) for carbaryl detection has been established. This sensitive (the limit of quantification (LOQ) was 2.0 × 10(-13) g mL(-1)), selective, simple and rapid method has been applied to determine trace carbaryl in water samples with the results consisting with those obtained by fluorimetry, showing its high accuracy. The apparent activation energy (E) and rate constant (k) of this activating reaction were 20.77 kJ mol(-1) and 1.85 × 10(-4) s(-1), respectively. Meanwhile, the mechanism of activating SSRTP for carbaryl detection was also discussed using infrared spectra (IR).

Long-term epilepsy surgery outcomes in patients with PET-positive, MRI-negative temporal lobe epilepsy.

This study compared the long-term efficacy of anterior temporal lobectomy (ATL) for the treatment of medically refractory temporal lobe epilepsy (TLE) in patients who presented with ipsilateral temporal PET hypometabolism and nonlesional magnetic resonance imaging (PET+/MRI-) with that in patients who had mesial temporal sclerosis (MTS) on MRI. We described the electroclinical, MRI, PET, and pathological characteristics and seizure outcome of 28 PET+/MRI--patients without discordant ictal and interictal electroencephalography (EEG) who underwent ATL (2004-2007) for medically refractory partial epilepsy while avoiding intracranial monitoring. The primary outcome was the percentages of Engel Class I outcomes at 2 and 5 years of PET+/MRI--patients compared with those of patients with MTS on MRI; neuropsychological testing was used as the secondary outcome. At 2-year follow-up, 21 (75%) patients in the PET+/MRI--group were in Engel Class I compared with 66 (75.9%) patients with MTS, and at 5-year follow-up, 20 (71.4%) patients in the PET+/MRI--group were in Engel Class I compared with 64 (73.6%) patients in the group with MTS. There were no significant differences between the groups at either time period. We concluded that normal MRI results should not preclude presurgical evaluations in patients with medically refractory TLE, as favorable long-term postoperative seizure outcomes are possible, especially in patients with unilateral anterior interictal epileptiform discharges and ipsilateral temporal PET hypometabolism.

Disconnective surgery in posterior quadrantic epilepsy: a series of 12 paediatric patients.

AIM: To assess the surgical outcomes of temporo-parieto-occipital (TPO) and parieto-occipital (PO) disconnection surgery for children with intractable posterior quadrantic epilepsy and a unilateral posterior quadrant lesion based on MRI and functional imaging abnormality in the TPO region on one side. METHODS: A retrospective review of data of 12 children who underwent TPO or PO disconnective surgery was carried out from September 2009 to September 2012. Three-dimensional surface reconstructions of MRI scans and intraoperative electrophysiological monitoring were used during surgery. Drugs were not discontinued after surgery in any patient. RESULTS: The affected hemisphere was the left in seven patients and the right in five patients. The mean ages at seizure onset and at surgery were four years and 12.3 years, respectively. At the time of surgery, 3 children had atonic seizures, 4 had symptomatic epilepsy with focal seizures and alteration of conscioussness, 4 had secondarily generalised seizures, and 1 child had spasms and tonic seizures. All patients had developmental delay. A pure TPO disconnection was performed in 11 patients and a PO disconnection was performed in the remaining patient. On pathological examination, 3 patients were shown to have focal cortical dysplasia (FCD) Ib, 2 with FCD IIa, 5 with FCD IIb, 1 with gliosis, and 1 with gliosis plus FCD IIa. Following surgery, 2 patients had oedema; 1 required another operation to resect the occipital lobe. At a mean follow-up of 34.5 months, 9 patients (75%) were classified as Engel class I, 2 as Engel Class II, and 1 as Engel class III. All 12 children had contralateral hemianopia postoperatively and improvement in median IQ (p=0.04) was reported three months postoperatively. CONCLUSIONS: With respect to the limits of a retrospective and relatively small sample size series TPO and PO disconnection are safe and effective motor-sparing epilepsy surgical procedures in selected patients with the epileptiform zone located in the posterior quadrant on one side.

Zr(H2O)2EDTA modulated luminescent carbon dots as fluorescent probes for fluoride detection.

A novel fluorescent probe (Zr(CDs-COO)(2)EDTA) has been designed for fluoride ion (F(-)) content detection based on the competitive ligand reactions carried out between the carboxylate groups (-COOH) on the surface of the luminescent carbon dots (CDs) and F(-) coordinated to Zr(H(2)O)(2)EDTA. The strong and stable fluorescence signal of this probe was quenched upon the addition of F(-) as a result of the formation of the non-fluorescent complex Zr(F)(2)EDTA, due to the stronger affinity of F(-) than the -COOH in the CDs to Zr(IV). The fluorescence change (ΔF) in this process was linear with respect to the content of F(-), ranging from 0.10 µM to 10 µM. The probe has been applied to F(-) detection in toothpaste and water samples with satisfactory results. Moreover, the mechanism of this Zr(H(2)O)(2)EDTA modulated fluorescent probe for the detection of F(-) was also discussed.

A new polysialic acid production process based on dual-stage pH control and fed-batch fermentation for higher yield and resulting high molecular weight product.

To determine the factors influencing the resulting molecular weight of polysialic acid (PSA), batch fermentations by using Escherichia coli were conducted. It was found that temperature and pH were significant factors affecting the PSA production and its resulting molecular weight. When pH was set at 6.4, temperature of 37 °C was suitable for cell growth and PSA production while 33 °C facilitated production of higher molecular weight of PSA. pH 6.4 was favorable for PSA production while pH 7.4 was good for higher molecular weight of PSA at 37 °C. Intramolecular self-cleavage of PSA might lead to relatively low molecular weight under mild acidic condition. Our data suggest that the PSA molecular weight is significantly affected by the pH condition rather than the temperature. It is concluded that the resulting PSA molecular weight not only depends on fermentation conditions but also relates to cell growth rate and PSA production rate. Higher PSA molecular weight was made when its production rate was faster than degradation rate. A novel two-stage pH control fermentation process for production of high molecular weight PSA was developed. At the first stage, pH was set at 6.4 to encourage cell growth and PSA production, whereas pH was set at 7.4 at the second stage to promote the formation of higher molecular weight PSA. PSA yield up to 5.65 g/L and its resulting molecular weight of 260 kDa was attained, the highest level ever reported.

A new effective process for production of curdlan oligosaccharides based on alkali-neutralization treatment and acid hydrolysis of curdlan particles in water suspension.

Biologically active ß-1,3-oligosaccharides with rapidly growing biomedical applications are produced from hydrolysis of curdlan polysaccharide. The water-insoluble curdlan impedes its hydrolysis efficiency which is enhanced by our newly developed alkali-neutralization treatment process to increase the stability of curdlan suspension to more than 20 days, while the untreated control settled within 5 min. A putative double-layer structure model comprising of a compact core and a hydrated outer layer was proposed to describe the treated curdlan particles based on sedimentation and scanning electron microscopy observation. This model was verified by single- and two-step acid hydrolysis, indicative of the reduced susceptibility to hydrolysis when close to the compact core. Electrospray ionization-mass spectrometry, thin-layer chromatography analyses, and effective HPLC procedure led to the development of improved process to produce purified individual ß-1,3-oligosaccharides with degrees of polymerization from 2 to 10 and potential for biomedical applications from curdlan hydrolyzate. Our new curdlan oligosaccharide production process offers an even better alternative to the previously published processes.

Catalytic solid substrate-room temperature phosphorimetry for the determination of residual perphenazine based on the electronic effect of rhodamine 6G.

The rhodamine 6G(+) -perphenazine (Rhod 6G(+) -PPH) compound is formed in the ester-exchange reaction between -OH of PPH and -COOC2 H5 of Rhod 6G(+) . PPH was oxidized to a red compound (PPH') in the presence of K2 S2 O8 . Interestingly, the room temperature phosphorescence (RTP) of Rhod 6G(+) was quenched because the -OH of PPH' reacted with -COOC2 H5 of Rhod 6G(+) -PPH to form Rhod 6G(+) -PPH' and PPH, which decreased the π-electron density (δ) of the carbon atom in the Rhod 6G(+) -PPH' conjugated system and enhanced the nonradiation energy loss of the excited Rhod 6G(+) of the triplet state. The PPH content was directly proportional to the ΔIp of the system. Thus, a new catalytic solid-substrate room temperature phosphorimetry (SSRTP) method was established for the determination of PPH. The method had high sensitivity (the limit of detection was 0.019 fg/spot, corresponding to a concentration of 4.8 × 10(-14) g/mL; the sampling quantity was 0.40 µL/spot), good selectivity, convenience and speed. The analytical results were in accordance with those of high-performance liquid chromatography (HPLC). The structures of Rhod 6G(+) , PPH and Rhod 6G(+) -PPH were characterized by infrared spectra. The reaction mechanism by which PPH was determined is discussed.

Determination of trace human chorionic gonadotropin by using multiwall carbon nanotubes as phosphorescence labeling reagent.

Taking advantage of the cutting effect of the strong oxidation of benzoyl peroxide [(C(6)H(5)CO)(2)O(2)] on the end of multiwall carbon nanotubes (MWNTs) to obtain water-soluble multiwall nanotubes (MWNTs') and the spiking effect of polyacrylamide (PA) on the room temperature phosphorescence (RTP) of MWNTs', a new phosphorescent labeling reagent, MWNTs'-PA, has been developed in this study. The product ß-Ab(HCG)-MWNTs'-PA obtained by MWNTs'-PA labeling human chorionic gonadotropin-ß-subunit three-dimensional core monoclonal antibody (ß-Ab(HCG)) not only could maintain good RTP characteristics of MWNTs' but also could take specific immunoreaction with ß-HCG to form ß-HCG- ß-Ab(HCG)-MWNTs'-PA, resulting in the increase of MWNTs' RTP signal. Thus, a new solid substrate room temperature phosphorescence immunoassay (SSRTPIA) for the determination of ß-HCG has been established. The limits of detection (LODs) of the new method were 0.021pgspot(-1) for the direct way at 447/615nm (λ(ex)(max)/λ(em)(max)) and 0.016pgspot(-1) for the sandwich way at 447/614nm (λ(ex)(max)/λ(em)(max)). This sensitive, accurate, and precise method was used to determine ß-HCG and diagnose human diseases by the direct way or the sandwich way, with the results coinciding with those obtained by chemiluminescence immunoassay. Meanwhile, the mechanisms of MWNTs' labeling ß-Ab(HCG) and determining ß-HCG are also discussed.

Selective determination of cysteine using BSA-stabilized gold nanoclusters with red emission.

Gold nanoclusters (AuNCs) were synthesized by a macromolecules template using bovine serum albumin (BSA) as stabilizer which can emit red photoluminescence under illumination of ultraviolet light. The fluorescence intensity of AuNCs enhanced through decreasing the surface defects of AuNCs modified with cysteine, herein we present a novel fluorometry for determination of trace cysteine. This method with a wider linear range from 2.0 to 800 nmol mL(-1), higher sensitivity (detection limit was 1.2 nmol mL(-1)) and better selectivity has been utilized to determine cysteine content in real samples, and the results were in a good agreement with those determined by electrochemical biosensor. At the same time, the structures of AuNCs and AuNCs-cysteine were characterized by Fourier-transform infrared spectroscopy (FTIR) and high resolution transmission electron microscopy (HRTEM) and the mechanism of the proposed assay for the detection of cysteine has been discussed.

Highly selective and sensitive detection of Cu2+ with lysine enhancing bovine serum albumin modified-carbon dots fluorescent probe.

Based on the ability of lysine (Lys) to enhance the fluorescence intensity of bovine serum albumin modified-carbon dots (CDs-BSA) to decrease surface defects and quench fluorescence of the CDs-BSA-Lys system in the presence of Cu(2+) under conditions of phosphate buffer (PBS, pH = 5.0) at 45 °C for 10 min, a sensitive Lys enhancing CDs-BSA fluorescent probe was designed. The environment-friendly, simple, rapid, selective and sensitive fluorescent probe has been utilized to detect Cu(2+) in hair and tap water samples and it achieved consistent results with those obtained by inductively coupled plasma mass spectroscopy (ICP-MS). The mechanism of the proposed assay for the detection of Cu(2+) is discussed.

Highly sensitive detection of residual chlorpromazine hydrochloride with solid substrate room temperature phosphorimetry.

Under the condition of 60 °C and 20 min at pH 6.12, chlorpromazine hydrochloride (CPZ) could react with fluorescein isothiocyanate (FITC) to produce FITC-CPZ, which increased the π-electron density (δ) of carbon atom in FITC conjugated system and the room temperature phosphorescence (RTP) intensity of FITC. Thus, a new solid substrate room temperature phosphorimetry (SSRTP) for the determination of residual CPZ was established. The regression equation of working curve was ΔI (p) = 4.254 + 7.906 m(CPZ) (ag spot(-1)) with the correlation coefficient (r) of 0.9990 in the range of 0.036-9.6 ag spot(-1) (corresponding concentration: 0.090-24 fg ml(-1), sample volume: 0.40 µl spot(-1)), and the detection limit (LD) was 0.018 ag spot(-1) (corresponding concentration: 4.5 × 10(-17) g ml(-1)). This method with wide linear range and high sensitivity was not only used to diagnose human disease based on the correlation between the residual quantity and lethal dose of CPZ in human serum, but also used to determine residual CPZ in biological samples with the results consisting with those obtained by gas chromatography (GC), showing good accuracy. The constituent of FITC-CPZ was analyzed by GC-MS (mass spectrometry) and the reaction mechanism of SSRTP for the determination of trace CPZ was also discussed.

Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of Agrobacterium sp. ATCC 31749.

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45-60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD(+), NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20-50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process.

Improvement of specific growth rate of Pichia pastoris for effective porcine interferon-α production with an on-line model-based glycerol feeding strategy.

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor. The influence of the glycerol feeding strategy on the specific growth rate and protein production was investigated. The traditional DO-stat feeding strategy led to very low cell growth rate resulting in low dry cell weight (DCW) of about 90 g/L during the subsequent induction phase. The previously reported Artificial Neural Network Pattern Recognition (ANNPR) model-based glycerol feeding strategy improved the cell density to 120 g DCW/L, while the specific growth rate decreased from 0.15 to 0.18 to 0.03-0.08 h(-1) during the last 10 h of the glycerol feeding stage leading to a variation of the porcine interferon-α production, as the glycerol feeding scheme had a significant effect on the induction phase. This problem was resolved by an improved ANNPR model-based feeding strategy to maintain the specific growth rate above 0.11 h(-1). With this feeding strategy, the pIFN-α concentration reached a level of 1.43 g/L, more than 1.5-fold higher than that obtained with the previously adopted feeding strategy. Our results showed that increasing the specific growth rate favored the target protein production and the glycerol feeding methods directly influenced the induction stage. Consequently, higher cell density and specific growth rate as well as effective porcine interferon-α production have been achieved by our novel glycerol feeding strategy.