Unoptimized Conditions Research Articles

Optimization of Extracellular Production of Recombinant Human Bone Morphogenetic Protein-7 (rhBMP-7) with Bacillus subtilis

Extracellular production of recombinant human bone morphogenetic protein-7 (rhBMP-7) was carried out through the fermentation of Bacillus subtilis. Three significant fermentation conditions and medium components were selected and optimized to enhance the rhBMP-7 production by using the response surface methodology (RSM). The optimum values of the three variables for the maximum extracellular production of rhBMP-7 were found to be 2.93 g/l starch, 5.18 g/l lactose, and a fermentation time of 34.57 h. The statistical optimization model was validated with a few fermentations of B. subtilis in shake flasks under optimized and unoptimized conditions. A 3-L jar fermenter using the shake-flask optimized conditions resulted in a higher production (413 pg/ml of culture medium) of rhBMP-7 than in a shake flask (289.1 pg/ml), which could be attributed to the pH being controlled at 6.0 and constant agitation of 400 rpm with aeration of 1 vvm.

Use of MALDI-ToF MS to elucidate the structure of oligomeric impurities formed during ‘click’ cyclization of polystyrene

Polymers with a cyclic topology exhibit a range of unique and potentially useful physical properties, including reduced rates of degradation and increased rates of diffusion in bulk relative to linear analogs. However, the synthesis of high purity cyclic polymers and verification of their structural purity remain challenging. The copper-catalyzed azide–alkyne “click” cyclization route toward cyclic polymers has been widely used, due to its synthetic ease and its compatibility with diverse polymer backbones. Yet unoptimized click cyclization conditions have been observed to generate oligomeric byproducts. In order to optimize these cyclization conditions, and to better understand the structure of the higher molecular weight oligomers, these impurities have been isolated and characterized. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI–ToF) MS is a particularly valuable characterization tool and was used to determine that the high molecular weight impurities are predominantly cyclic oligomers. It should also be noted that the rapid analysis and small analyte requirements of this MS technique make it particularly attractive as a general tool for elucidating polymer architecture.

Optimization Studies for Enhancing Cellulase Production by Penicillium janthinellum Mutant EU2D-21 Using Response Surface Methodology

Extracellular fungal cellulases are key enzymes for the degradation of lignocellulosic biomass. Greater production of these enzymes could reduce the cost of biofuels production. In this study, the basal medium for cellulase production by a Penicillium janthinellum mutant (EU2D-21) in submerged fermentation conditions was optimized using response surface methodology (RSM). Initial studies using a Plackett-Burman design (PBD) showed that (NH4)2SO4 and urea are significant factors for improving β-glucosidase and FPase production. A central composite design (CCD) was applied to obtain the maximum response, which resulted in the optimal production of β-glucosidase (5.79 IU/mL) and FPase (5.76 IU/mL). These values were 1.87 and 1.67 times higher than the corresponding values obtained under un-optimized conditions.

Optimization of extracellular chromate reductase production by Bacillus amyloliquefaciens (CSB 9) isolated from chromite mine environment

Abstract Chromate reductase production by a bacterial isolate CSB 9 has been optimized to a high titer in the present investigation. The bacterium isolated previously from a chromium contaminated site and identified as Bacillus amyloliquefaciens following biochemical and 16S rRNA gene sequencing, was evaluated for chromate reductase production. Chromate reductase activity of B. amyloliquefaciens (CSB 9) was associated with the contribution of extracellular enzymes under the influence of various physico-chemical parameters. In presence of 50 µM of Cr(VI) concentration, the bacterial growth exhibited 8 h of lag phase, 8–32 h of log phase and stationary phase upto 48 h. The maximum chromate reductase production was achieved in the presence of 50 µM of Cr(VI); 0.5% (w/v) of glucose; 1% (w/v) of trypton; 0.5% (w/v) of Na 2 HPO 4 ; 0.5% (w/v) KNO 3 ; 0.5% (w/v) KCl and 0.5% (w/v) thiamine as source of vitamin with the initial medium pH of 7.0 and incubation at 35 °C under shaking condition (100 rpm). In optimized conditions, the chromate reductase production was enhanced to a significant level of 3.67±0.014 U ml −1 as compared to unoptimized condition (2.37±0.029 U ml −1 ) with decrease in time from 20 h (late log phase) under unoptimized condition to 16 h under optimized condition. This communication elucidates the enhanced production of extracellular chromate reductase from a newly isolated bacterium B. amyloliquefaciens from chromite mine soil under optimized conditions which can be potentially exploited for biotechnological application.

Isolation, characterization, and validation of oleaginous, multi-trophic, and haloalkaline-tolerant microalgae for two-stage cultivation

Mass outdoor cultivation of microalgae for biofuels and co-products faces challenges of low lipid productivity, contamination, inefficient CO2 supply, and difficulties in harvesting. A two-stage cultivation process was developed to address these challenges. This involves culturing microalgae in a fermentor heterotrophically or photobioreactor mixotrophically as first-stage to rapidly obtain high cell densities for inoculating a phototrophic open-pond culture featuring high levels of NaHCO3, pH, and salinity as second-stage. A microalgae that is tolerant of these phototrophic conditions, can use organic carbon, and can prolifically produce oil is key to the success of such a two-stage process. Two oleaginous, haloalkaline-tolerant, and multi-trophic green microalgae from soda lakes were isolated, identified, and compared in this study using a multi-instrument approach as candidates for such process. A model triacylglyceride (TAG) was developed for rapid, non-destructive lipid quantitation using liquid-state 1H NMR. A two-stage cultivation system and a high pH-mediated auto-flocculation method were tested on the selected strain ALP2 with a 1L fermentor and 40L open-tank. In unoptimized conditions, the stain achieved a final biomass concentration of 0.978gDCW/L, lipid content of 39.78% DCW, and auto-flocculation harvesting efficiency of 64.1%.

Application of statistical experimental design for optimization of silver nanoparticles biosynthesis by a nanofactory Streptomyces viridochromogenes

Central composite design was chosen to determine the combined effects of four process variables (AgNO3 concentration, incubation period, pH level and inoculum size) on the extracellular biosynthesis of silver nanoparticles (AgNPs) by Streptomyces viridochromogenes. Statistical analysis of the results showed that incubation period, initial pH level and inoculum size had significant effects (P<0.05) on the biosynthesis of silver nanoparticles at their individual level. The maximum biosynthesis of silver nanoparticles was achieved at a concentration of 0.5% (v/v) of 1 mM AgNO3, incubation period of 96 h, initial pH of 9 and inoculum size of 2% (v/v). After optimization, the biosynthesis of silver nanoparticles was improved by approximately 5-fold as compared to that of the unoptimized conditions. The synthetic process of silver nanoparticle generation using the reduction of aqueous Ag+ ion by the culture supernatants of S. viridochromogenes was quite fast, and silver nanoparticles were formed immediately by the addition of AgNO3 solution (1 mM) to the cell-free supernatant. Initial characterization of silver nanoparticles was performed by visual observation of color change from yellow to intense brown color. UV-visible spectrophotometry for measuring surface plasmon resonance showed a single absorption peak at 400 nm, which confirmed the presence of silver nanoparticles. Fourier Transform Infrared Spectroscopy analysis provided evidence for proteins as possible reducing and capping agents for stabilizing the nanoparticles. Transmission Electron Microscopy revealed the extracellular formation of spherical silver nanoparticles in the size range of 2.15-7.27 nm. Compared to the cell-free supernatant, the biosynthesized AgNPs revealed superior antimicrobial activity against Gram-negative, Gram-positive bacterial strains and Candida albicans.

Kinetic modeling, production and characterization of an acidic lipase produced by Enterococcus durans NCIM5427 from fish waste.

Enterococcus durans NCIM5427 (ED-27), capable of producing an intracellular acid stable lipase, was isolated from fish processing waste. Its growth and subsequent lipase production was optimized by Box Behneken design (optimized conditions: 5%v/v fish waste oil (FWO), 0.10mg/ml fish waste protein hydrolysates (FWPH) at 48h of fermentation time). Under optimized conditions, ED-27 showed a 3.0 fold increase (207.6U/ml to 612.53U/ml) in lipase production, as compared to un-optimized conditions. Cell growth and lipase production was modeled using Logistic and Luedeking-Piret model, respectively; and lipase production by ED-27 was found to be growth-associated. Lipase produced by ED-27 showed stability at low pH ranges from 2 to 5 with its optimal activity at 30°C , pH4.6; showed metal ion dependent activity wherein its catalytic activity was activated by barium, sodium, lithium and potassium (10mM); reduced by calcium and magnesium (10mM). However, iron and mercury (5mM) completely inactivated the enzyme. In addition, modifying agents like SDS, DTT, β-ME (1%v/v) increased activity of lipase of ED-27; while, PMSF, DEPC and ascorbic acid resulted in a marked decrease. ED-27 had maximum cell growth of 9.90309log CFU/ml under optimized conditions as compared to 13log CFU/ml in MRS. The lipase produced has potential application in poultry and slaughterhouse waste management.

Extracellular endo-mannanase from Bacillus sp. CFR1601: Economical production using response surface methodology and downstream processing using aqueous two phase system

Bacillus sp. CFR1601, isolated from decaying plant litter, produced an extra-cellular endo-mannanase (198.0IU/g) under solid state fermentation (SSF) using defatted coconut residue as the prime solid substrate. In order to enhance endo-mannanase production, three component, five level central composite design (CCD) of response surface methodology (RSM) was used. Based on contour plots and variance analysis, optimum conditions for endo-mannanase production from Bacillus sp. CFR1601 were attained when defatted coconut residue was supplemented with sesame oil meal (10.0, w/w), Tween-80 (0.2%, v/v) and inoculated with bacterial cells from log phase (12h old; OD600nm≈3.6). The empirical model developed through RSM brought about 4.04–4.39-fold (800.0–870.0IU/g) improvement in endo-mannanase yield as compared to un-optimized growth conditions. Downstream processing of endo-mannanase from SSF media was carried out for the first time using polyethylene glycol (PEG)/salt aqueous two phase system (ATPS). ATPS system consisting of a combination of PEG 3350 12.0% (w/w), Na2SO4 12.0% (w/w), protein load 10.0% (w/w) and pH 5.0 resulted in one-sided partitioning of endo-mannanase towards bottom phase with 3.8-fold purification and 95.4% recovery. Second stage ATPS with fresh top phase further improved purification of endo-mannanase to 12.32-fold. Our overall results suggest a cost-effective and integrated process for production and downstream processing of endo-mannanase.

Statistical approach to study the interactive effects of process parameters for enhanced xylitol production by Candida tropicalis and its potential for the synthesis of xylitol monoesters

Previous results showed that an indigenously isolated yeast strain of Candida tropicalis was found to produce 12.11 g/L of xylitol under unoptimized conditions in presence of 50 g/L of xylose. In the present study, optimizing the process using one-variable at-a-time resulted in the production of 59.07 g/L of xylitol in 96 h in presence of 100 g/L xylose. Further optimization using response surface methodology led to the production of 65.45 g/L in medium containing 100 g/L xylose, 0.5% yeast extract, 0.03% MgSO(4).7H(2)O and 0.2% KH(2)PO(4), pH-4.5, 30 °C, 200 r/min for 96 h with 4% inoculum level. Addition of 1% methanol in response surface methodology optimized-medium led to the production of 67.12 g/L. Scaling up in 10 L fermentor resulted in productivity of 0.80 g/Lh with yield of 0.68 g/g. Efficient synthesis of xylitol esters was achieved with butyric acid (50.32%) and caproic acid (38.36%) in 4 h using Pseudomonas aeruginosa lipase in t-butanol: tetrahydrofuran (1:1 v/v).

Fermentative production of shikimic acid: a paradigm shift of production concept from plant route to microbial route

Different physiological and nutritional parameters affect the fermentative production of shikimic acid. In our study, Citrobacter freundii initially produced 0.62 g/L of shikimic acid in 72 h. However, when process optimization was employed, 5.11 g/L of shikimic acid was produced in the production medium consisting of glucose (5.0 %), asparagine (4.5 %), CaCO3 (2.0 %), at pH 6.0, when inoculated with 6 % inoculum and incubated at 30 ± 1 °C, 200 rpm for 60 h. Preliminary fed-batch studies have resulted in the production of 9.11 g/L of shikimic acid on feeding the production medium by 20 g/L of glucose at 24 h of the fermentation run. Production of similar amount of shikimic acid was observed when the optimized conditions were employed in a 10-L bioreactor as obtained in shake flask conditions. A total of 9.11 g/L of shikimic acid was produced in 60 h. This is approximately 14.69-fold increase in shikimic acid production when compared to the initial un-optimized production conditions. This has also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.

Increased alkalotolerant and thermostable ribonuclease (RNase) production from alkaliphilic Streptomyces sp. M49-1 by optimizing the growth conditions using response surface methodology

Total of 171 alkaliphilic actinomycetes were evaluated for extracellular RNase production and Streptomyces sp. M49-1 was selected for further experiments. Fermentation optimization for RNase production was implemented in two steps using response surface methodology with central composite design. In the first step, the effect of independent fermentation variables including temperature, initial pH and process time were investigated. After identification of carbon and nitrogen sources affecting the production by one variable at a time method, concentrations of glucose and yeast extract and also inoculum size were chosen for the second central composite design. A maximum RNase activity was obtained under optimal conditions of 4.14 % glucose concentration, 4.63 % yeast extract concentration, 6.7 × 10⁶ spores as inoculum size for 50 ml medium, 42.9 °C, 91.2 h process time and medium initial pH 9.0. Optimum activity of the enzyme is achieved at pH 11 and temperature 60 °C. The enzyme is highly stable at pH range 9.0-12.0 and at 90 °C after 2 h. Statistical optimization experiments provide 2.25 fold increases in the activity of alkalotolerant and thermostable RNase and shortened the fermentation time compared to that of unoptimized condition. The members of Streptomyces can be promising qualified RNase producer for pharmaceutical industries.

Optimization of extracellular lipase production by Debaryomyces hansenii isolates from dry-salted olives using response surface methodology

The lipase production by Debaryomyces hansenii strains isolated from dry-salted olives cv. Thassos was investigated. Glucose, olive oil and pH were essential to obtain a high lipase yield. Optimization of the medium components which enhance lipase production by the strain D. hansenii YLL29 was achieved with the aid of response surface methodology. The composition of the optimized medium to enhance lipase production by D. hansenii is as follows (g/L): yeast extract 5.0, peptone 10, K2HPO4 4.0, MgSO4·7H2O 1.0, glucose 13.1, olive oil 19, Tween 80 3.8, and pH 6.4. Practical validation of the optimum medium gave lipase activity 7.44U/mL, which was 2.28-fold higher than the unoptimized conditions. Under the optimized conditions the twenty D. hansenii isolates showed increased lipase activity fluctuating between 6.00 and 7.44U/mL. The results corroborated the validity and the effectiveness of the model, as the statistical approaches proved to be suitable in predicting the optimum production medium composition for maximum extracellular lipase yield. The high lipolytic activity of D. hansenii YLL29 (7.44U/mL) indicates the possible commercial importance of this isolate.

Middle-redox potential laccase from Ganoderma sp.: its application in improvement of feed for monogastric animals

The variables influencing laccase production by white-rot fungus Ganoderma sp. rckk-02 were optimized employing response surface methodology. Malt extract (6.0% w/v), lignin (0.5% w/v) and pH (5.5) were found to be the most significant factors for enhanced laccase production by 7 fold (226.0 U/ml) as compared to unoptimized growth conditions (32.0 U/ml). The N-terminal sequence of laccase revealed its distinct amino acid profile (S- I- R- N- S- G), which suggested it as a novel enzyme. The Far-UV CD spectrum of the laccase showed single broad negative trough at around 213 nm, a typical signature of all β proteins. The laccase was found to fall in the range of middle redox potential laccases. Purified laccase at dosage of 2.5 Ug−1 body weight when supplemented with pelleted diet of rats, a significant improvement (p < 0.05) in nutrients digestibility without causing any elevation of blood stress enzymes was observed.

Optimization of process variables for the synthesis of silver nanoparticles by Pycnoporus sanguineus using statistical experimental design

Sequential optimization strategy based on statistical experimental design and one-factor-at-a-time (OFAT) method were employed to optimize the process parameters for the enhancement of silver nanoparticles (AgNPs) production through biological synthesis using Pycnoporus sanguineus. Based on the OFAT method, three significant components influencing the size of AgNPs produced were identified as AgNO3 concentration, incubation temperature, and agitation speed. The optimum values of these process parameter for the synthesis of AgNPs were determined using response surface methodology (RSM) based on Box-Behnken design. The validity of the model developed was verified, and the statistical analysis showed that the optimum operating conditions were 0.001 M of AgNO3, 38°C, and 200 rpm with the smallest AgNPs produced at 14.86 nm. The disc diffusion method also suggested that AgNPs produced using optimum conditions have higher antimicrobial activity compared to the unoptimized AgNPs. The present study developed a robust operating condition for the production of AgNPs by P. sanguineus, which was 8.6-fold smaller than that obtained from un-optimized conditions.

From crude glycerol to carotenoids by using a Rhodotorula glutinis mutant

In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280% increase in β-carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R(2)=0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P<0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07±1.45mgl(-1)) under optimized growth conditions was not statistically different from the maximal predicted (14.64±1.57mgl(-1)) (P<0.05), and it was about 100% higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.

헥사플루오르프로필렌 올리고머를 사용한 헥사플루오르프로필렌 옥사이드의 음이온 중합

Hexafluoropropylene(HFP) dimer와 trimer로 구성된 올리고머의 조성비, 반응온도 그리고 hexafluoropropylene oxide(HFPO) 투입속도가 HFPO 음이온 중합반응에 미치는 영향 등을 알아보았다. HFP 올리고머는 불화금속 CsF와 TG를 사용한 음이온 반응을 통해 합성하였고, CsF 5 g, TG 10 g 그리고 반응온도 <TEX>$0^{\circ}C$</TEX>에서 합성된 HFP 올리고머의 dimer 함량은 상대적으로 높은 35.1%를 나타내었다. HFPO 음이온 중합의 경우, 반응온도 <TEX>$0^{\circ}C$</TEX>, HFP dimer 35.1%를 포함하고 있는 올리고머와 HFPO 투입속도 1.85 g/min에서 Cs(HFPO)n 알콕사이드는 상대적으로 원활한 사슬성장을 하면서 중량평균 분자량 3600을 나타내었다. 반면, 반응온도 <TEX>$10^{\circ}C$</TEX> 및 증가된 HFPO 투입속도에서는 알콕사이드의 <TEX>$F^{{\delta}-}$</TEX>의 이탈현상을 촉진시켜 중평균 분자량은 감소되었다. 결론적으로, HFP 올리고머를 용매로 사용한 HFPO 음이온 중합반응의 사슬성장 및 사슬전이는 용매의 조성, 반응온도 및 단량체의 투입속도에 영향을 받고 있음을 알 수 있었다. Anionic polymerization of hexafluoropropylene oxide (HFPO) was investigated under various reaction conditions such as various hexafluoropropylene (HFP) oligomers composed of dimer and trimer, reaction temperatures, and feeding rates of hexafluoropropylene oxide monomer. HFP oligomer was synthesized from cesium fluoride (CsF) and HFP in tetraethyleneglycol dimethylether (TG). Under 5 g of CsF, 200 g of HFP, 10 g of TG, and reaction temperature <TEX>$30^{\circ}C$</TEX>, HFP dimer content in oligomer was relatively increased. HFPO oligomer with a high molecular weight (<TEX>$M_w$</TEX> 3600) was synthesized in conditions of reaction temperature <TEX>$0^{\circ}C$</TEX>, HFP oligomer with 35.1% of dimer, and 1.85 g/min of HFPO feeding rate. Otherwise, chain transfer was increased under unoptimized reaction conditions. Consequently, it was found that reaction conditions impact chain propagation and chain transfer in the anionic polymerization of HFPO.

Optimization of Submerged Fermentation Conditions for Lovastatin Production by the Culinary-Medicinal Oyster Mushroom, Pleurotus ostreatus (Higher Basidiomycetes)

Statistical experimental designs were used to optimize lovastatin production by culinary-medicinal oyster mushroom Pleurotus ostreatus OBCC 1031 under submerged fermentation. The Plackett-Burman design was used to determine effective culture parameters, glucose, lactose, maltose, glycerol, peptone, yeast extract, NH4SO2, NaCl, thiamine, and agitation speed. Statistical analyses of data from the Plackett-Burman design show that glucose, yeast extract, and agitation speed are significant parameters. The interactive effects of these culture parameters on lovastatin production by P. ostreatus OBCC 1031 were further studied by a Box-Behnken design. Maximum lovastatin production (114.82 mg/L) was reached after 6 days of fermentation in optimized culture conditions (30 g/L glucose, 10 g/L yeast extract, 200 rpm, 28°C, and pH 6). This amount was found to be 50 times higher than that produced under unoptimized conditions in submerged fermentation by P. ostreatus.

Constitutive Optimized Production of Streptokinase inSaccharomyces cerevisiaeUtilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter ofPichia pastoris

A novel expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay and in vitro clot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions.

STATISTICAL OPTIMIZATION OF GREEN FLUORESCENT PROTEIN PRODUCTION FROM Escherichia coli BL21(DE3)

An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box–Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD600nm) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.

Fermentation optimization for the production of lipid by Cryptococcus curvatus: Use of response surface methodology

Cryptococcus curvatus is able to grow on crude glycerol derived from the biodiesel production process. Through Box–Behnken design and response surface methodology, the optimal temperature, pH, and glycerol concentration for yeast growth and lipid production on pretreated crude glycerol was identified as 30.2 °C, 6.0, and 19.8 g/l, respectively. Adopting these optimal parameters, the biomass density obtained was 7.11 ± 0.36 g/l with a lipid content of 38.53 ± 1.88%, which matched well with predicted values of 6.98 g/l and 41.31%, respectively. The resulting parameters of the response surface method optimization were used in a fed-batch fermentation where crude glycerol was automatically pumped in responding to exhausted oxygen levels in the fermentor. At the end of 12 days, the biomass concentration was 44.53 g/l and the lipid content was 49.0%. Compared with our previous fed-batch experiment which was conducted under un-optimized condition and manual feeding, the yield of biomass and lipid increased 35.26% and 25.29%, respectively. The optimal parameters and the automatic pumping scheme developed from this study will assist greatly in future industrial scale application of producing microbial oils from crude glycerol.