Optimization of Chlorella vulgaris cultivation grown in waste molasses syrup using mixture design

The aim of this study was to determine and optimize culture media for Chlorella vulgaris microalgae under mixotrophic conditions using waste molasses as a cheap carbon source containing both organic carbons and other nutrients. In the current study, at first the growth and lipid productivity of C. vulgaris were assessed in different culture media and the best media was selected for mixotrophic growth conditions. Significant medium ingredients were screened through Plackett–Burman design. Then ingredients with positive effect were considered as a mixture component and their combinations were evaluated on lipid productivity using mixture design. According to results, Zarrouk medium was considered as the base medium with the highest biomass and lipid productivity of 72 and 7.1 mg L⁻¹ d⁻¹, respectively. Based on the Plackett–Burman design, out of 11 factors, molasses, NaNO₃ and K₂HPO₄ demonstrated key roles in biomass and lipid productivity in mixotrophic conditions. Consequently, the selected three factors were investigated by mixture design. The results showed that high concentration of molasses causes decrease in biomass and lipid productivity due to high turbidity and a blend consisting of approximately 9.5 g L⁻¹ molasses, 5 g L⁻¹ NaNO₃ and 0.15 g L⁻¹ K₂HPO₄ was found as the optimum mixture with obtained lipid productivity of 115 mg L⁻¹ d⁻¹. In conclusion, waste molasses can be used as a promising feedstock for cost effective cultivation of C. vulgaris.     

Fermentation strategy for the production of poly(3-hydroxyhexanoate) by <Emphasis Type="Italic">Aeromonas sp.</Emphasis> KC014

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) was produced by Aeromonas sp. KC014 strain isolated from Taiwan in soil environment in the flask and fermentor cultures. The medium optimization, such as carbon source and nitrogen source, carbon-nitrogen ratio was conducted to obtain the optimum 3-hydroxyhexanoate content. The defined medium with dodecanoic acid as the carbon source and (NH₄)₂SO₄ as the nitrogen source was obtained as the main medium. When cells grown in medium containing 30 g/L dodecanoic acid, 15 g/L sodium gluconate and 1 g/L soytone (C/N was 30/1) as final PHA concentration, the cell dry weight and HB content of 5.16 g/L, 14.0 g/L and 36.0%, respectively, were obtained. The maximum HHx/PHA content increased from 0.1% to 1.3% nearly 12-fold when the dissolved oxygen was decreased from 40% to 20%. P(3HB-co-3HHx) biosynthesis was triggered by the addition of limited nitrogen, phosphorus and magnesium to get a maximum HHx/PHA content of 14% in 95 hours. This work was presented at 13 ^th YABEC symposium held at Seoul, Korea, October 20–22, 2007.     

Revealing the Phenotypic and Genomic Background for PHA Production from Rapeseed-Biodiesel Crude Glycerol Using Photobacterium ganghwense C2.2

Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain’s metabolic capabilities is necessary in choosing economically feasible production conditions. This study aimed to create an in-depth view of the utilization of Photobacterium ganghwense C2.2 for PHA production by linking a wide array of characterization methods: metabolic pathway annotation from the strain’s complete genome, high-throughput phenotypic tests, and biomass analyses through plate-based assays and flask and bioreactor cultivations. We confirmed, in PHA production conditions, urea catabolization, fatty acid degradation and synthesis, and high pH variation and osmotic stress tolerance. With urea as a nitrogen source, pure and rapeseed-biodiesel crude glycerol were analyzed comparatively as carbon sources for fermentation at 20 °C. Flask cultivations yielded 2.2 g/L and 2 g/L PHA at 120 h, respectively, with molecular weights of 428,629 g/mol and 81,515 g/mol. Bioreactor batch cultivation doubled biomass accumulation (10 g/L and 13.2 g/L) in 48 h, with a PHA productivity of 0.133 g/(L·h) and 0.05 g/(L·h). Thus, phenotypic and genomic analyses determined the successful use of Photobacterium ganghwense C2.2 for PHA production using urea and crude glycerol and 20 g/L NaCl, without pH adjustment, providing the basis for a viable fermentation process.     

Utilization of response surface methodology to optimize the culture media for the production of rhamnolipids by Pseudomonas aeruginosa AT10

Pseudomonas aeruginosa AT10 produced a mixture of surface‐active rhamnolipids when cultivated on mineral medium with waste free fatty acids as carbon source. The development of the production process to an industrial scale included the design of the culture medium. A 2⁴ full factorial, central composite rotational design and response surface modelling method (RSM) was used to enhance rhamnolipid production by Pseudomonas aeruginosa AT10. The components that are critical for the process medium were the carbon source, the nitrogen source (NaNO₃), the phosphate content (K₂ HPO₄/KH₂PO₄ 2:1) and the iron content (FeSO₄·7H₂O). Two responses were measured, biomass and rhamnolipid production. The maximum biomass obtained was 12.06 g dm^?3 DCW, when the medium contained 50 g dm^?3 carbon source, 9 g dm^?3 NaNO₃, 7 g dm^?3 phosphate and 13.7 mg dm^?3 FeSO₄·7H₂O. The maximum concentration of rhamnolipid, 18.7 g dm^?3, was attained in medium that contained 50 g dm^?3 carbon source, 4.6 g dm^?3 NaNO₃, 1 g dm^?3 phosphate and 7.4 mg dm^?3 FeSO₄·7H₂O. © 2002 Society of Chemical Industry     

Enhanced production of l(+)-lactic acid by floc-form culture of Rhizopus oryzae

A process to optimize l-lactic acid production from glucose by Rhizopus oryzae, based on sustaining floc morphology throughout the fermentation process, is herein performed. During the fermentation, supplementary ammonium sulfate was added intermittently to maintain the ammonia level of the culture medium always higher than 0.1 g/L. With replenish of nitrogen source, mycelia flocs did not aggregate, and the lactic acid production was optimized upon the fermentation being controlled at pH 4.3–4.5 by adding calcium carbonate slurry. In contrast, without supplementary addition of nitrogen source, mycelial clumps formed, resulting in a poor production of lactic acid. In the initial batch fermentation process, the final concentration of lactic acid produced was 109 g/L, with a yield (g lactic acid/g glucose consumed) of 0.87 and a productivity of 2.73 g/L h, using 125 g/L of glucose as substrate. For the first four cycles of repeated-batch fermentation, the average final concentration, the productivity and the yield of lactic acid were 113 g/L, 4.03 g/L h and 0.90, respectively.     

Characteristics of hydrogen production by immobilized cyanobacterium Microcystis aeruginosa through cycles of photosynthesis and anaerobic incubation

The characteristics of hydrogen production using immobilized cyanobacterium Microcystis aeruginosa were studied through a two-stage cyclic process. Immobilized cells were first grown photosynthetically under CO₂ and light, followed by anaerobic H₂ production in the absence of light and sulfur. M. aeruginosa was capable of generating H₂ under immobilized conditions, and the use of immobilized cells allowed the maintenance of stable production and sped up the changes in culture conditions for cyclic two-stage operation. M. aeruginosa was also capable of utilizing exogenous glucose as a substrate to generate hydrogen and 30 mM concentration proved to be optimal. The externally added glucose improved H₂ production rates, total produced volume and the lag time required for cell adaptation prior to H₂ evolution. The rate of hydrogen evolution was increased as temperature increased, and the maximum evolution rate was 48 mL/h/L and 34.0 mL/h/L at 42 °C and 37 °C, respectively. The optimal temperature for hydrogen production was 37–40 °C because temperatures higher than 42 °C resulted in cell death. In order to continue repeated cycles of H₂ production, at least two days of photosynthesis under conditions with light, CO₂, and sulfur should be allowed for cells to recover H₂ production potential and cell viability.     

Production of poly‐3‐hydroxybutyrate by Cupriavidus necator from corn syrup: statistical modeling and optimization of biomass yield and volumetric productivity

BACKGROUND: The paper reports an investigation into the possibility of producing poly‐3‐hydroxybutyrate (P(3HB)) polyester using corn syrup, a relatively low cost by‐product from the starch industries. The concentrations of medium components, corn syrup, dipotassium hydrogen phosphate (K₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄) and ammonium sulfate [(NH₄)₂SO₄] were optimized using design of experiments (DOE). RESULTS: Response surface methodology (RSM) under central composite face design (CCFD) was used to obtain the optimum values of medium components and responses in terms of biomass yield and volumetric P(3HB) productivity. The highest P(3HB) productivity and biomass yield obtained were 0.224 g L^?1 h^?1 and 0.57 g g^?1, respectively. A limited‐nitrogen concentration had a higher volumetric P(3HB) productivity (0.170 g L^?1 h^?1) than that of the excess nitrogen batch experiment (0.0675 g L^?1 h^?1). The optimum corn syrup:N:P ratio of 50:0.078:1 was based on numerical optimization of the desirability function between biomass yield and volumetric P(3HB) productivity by Cupriavidus necator DSMZ 545. CONCLUSION: The results obtained in this study demonstrated that P(3HB) could be efficiently produced to a high concentration with high productivity by applying nitrogen limitation in a defined medium, indicating this agricultural by‐product to be a suitable nutrient source in further studies to develop biomaterials through biotechnology. Copyright © 2010 Society of Chemical Industry     

Optimization of Rhamnolipid Production by <Emphasis Type="Italic">P. aeruginosa</Emphasis> Isolate P6

Rhamnolipids are interesting microbial surfactants having great industrial importance. However, the main obstacles towards an economic production of rhamnolipids are low productivity and high raw-material costs. Therefore, this study aimed at optimization of the culture media as well as culture conditions using response surface methodology for maximum rhamnolipid production by Pseudomonas aeruginosa isolate P6, a promising rhamnolipid-producing isolate. The optimum medium for maximum rhamnolipid production was found to be a mineral salts medium with glycerol 2 % v/v as the carbon source. The optimum cultivation conditions using response surface methodology were found to be an incubation temperature of 30 °C, an agitation rate of 250 rpm, an inoculum size of 5 % v/v and unlike most studies, an initial pH of 7.5. The resulting model predicted data points that corresponded well to the experimental values. Optimization resulted in a threefold increase in rhamnolipid production reaching 7.54 g/L. The data are potentially useful for further industrial exploitation of rhamnolipid production by the studied isolate.     

Efficient Utilization of Crude Glycerol as Fermentation Substrate in the Synthesis of Poly(3-hydroxybutyrate) Biopolymers

One refined and two crude glycerol (from biodiesel production) samples were utilized to produce poly(3-hydroxybutyrate) (PHB) by Pseudomonas oleovorans NRRL B-14682. A batch culture fermentation protocol including 1% glycerol and an aeration rate of 3 standard liters per minute proved best for PHB synthesis (av. yield = 1.0 ± 0.2 g/L at 48 h) and efficient glycerol utilization. PHB molecular weights decreased as MeOH concentration increased. Refined glycerol resulted in PHB polymers with number average molecular weights (M _n) of 314,000 g/mol which decreased by 17 and 90% as MeOH media concentrations increased to <0.005 and 0.85%, respectively. Proton (¹H) NMR demonstrated the presence of glycerol- and methoxy-based end-capping, which was confirmed by ¹H diffusion experiments (DOSY analyses). NMR diffusion analyses of the PHB polymers established their diffusivities, and confirmed that their relative molecular sizes were dependent on the impurities in the glycerol. In addition, DOSY analyses indicated that each end-capped PHB polymer and the glycerol or methoxy groups bound to it had the same diffusion constants, demonstrating that they migrated together as covalent complexes. Non-covalent complexation was eliminated by physically mixing free glycerol with PHB synthesized from oleic acid; their respective diffusivities were notably faster.     

Influence of Temperature on Growth and Peak Oil Biosynthesis in a Carbon-Limited Medium by <Emphasis Type="Italic">Pythium irregulare</Emphasis>

Kinetic analysis was investigated for a carbon-limited medium (C/N ratio = 5.0) supporting the growth of the 5,8,11,14,17-eicosapentaenoic acid (20:5; ω-3) (EPA)-accumulating fungal organism Pythium irregulare. The productivity and yield parameters at three temperatures, 14, 21, and 28°C, demonstrated growth-coupled synthesis for lipid-free biomass growth and lipid accumulation. For this system, the maximum specific growth rate and theoretical maximum biomass yield based on logistic growth kinetics were used to determine an activation energy of the growth process, E _g, of 36.5 kJ mol⁻¹. At 14, 21, and 28°C, peak lipid yield occurred after culturing for 7, 4, and 3 days, respectively, with peak lipid yields of 8.14, 12.8, and 6.69 g lipid 100 g⁻¹ glucose. At these peak yields, the maximum lipid-free biomass productivity was achieved at the colder 14°C temperature as well as an increased concentration of EPA—10.9 wt%. Despite these enhancements, the maximum relative lipid production (P _R(FA/B)) was achieved at 21°C—19.1%.     

Batch production of L(+) lactic acid from whey by Lactobacillus casei (NRRL B‐441)

The effects of temperature, pH, and medium composition on lactic acid production by Lactobacillus casei were investigated. The highest lactic acid productivity values were obtained at 37 °C and pH 5.5. The productivity was 1.87 g dm^?3 h^?1 at 37 °C in shake flasks. In the fermenter, a productivity of 3.97 g dm^?3 h^?1 was obtained at pH 5.5. The most appropriate yeast extract concentration was 5.0 g dm^?3. Whey yielded a higher productivity value than the analytical lactose and glucose. Initial whey lactose concentration did not affect lactic acid productivity. MnSO₄ ·H₂O was necessary for lactic acid production by L casei from whey. Product yields were approximately 0.93 g lactic acid g lactose^?1. Copyright © 2004 Society of Chemical Industry     

Medium optimization for lipid production through co‐fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi

Co‐fermentation of lignocellulose‐based carbohydrates is a potential solution to improve the economics of microbial lipid production. In the present paper, experiments were performed to optimize the media composition for lipid production by the oleaginous yeast Lipomyces starkeyi AS 2.1560 through co‐fermentation of glucose and xylose (2 : 1 wt/wt). Statistical screening of nine media variables was performed by a Plackett–Burman design. Three factors, namely mixed sugar, yeast extract and FeSO₄, were found as significant components influencing cellular lipid accumulation. Further optimization was carried out using a Box–Behnken factorial design to study the effects of these three variables on lipid production. A mathematical model with the R² value at 96.66% was developed to show the effect of each medium composition and their interactions on the lipid production. The model estimated that a maximal lipid content of 61.0 wt‐% could be obtained when the concentrations of mixed sugar, yeast extract and FeSO₄ were at 73.3 g/L (glucose 48.9 g/L, xylose 24.4 g/L), 7.9 g/L and 4.0 mg/L, respectively. The predicted value was in good accordance with the experimental data of 61.5%. Compared with the initial media, the optimized media gave 1.59‐fold and 2.03‐fold increases for lipid content and lipid productivity, respectively.     

Biological effects of poly(ethylene glycol) on the microbial poly(β-hydroxyalkanoates) produced by pseudomonads microorganisms

Poly(ethylene glycol) of M_n 200 g/mol (PEG-200) was added to cultivation media of Pseudomonas oleovorans and Pseudomonas putida during fermentation. The carbon source and the medium used were sodium octanoate and medium E*, respectively. It was discovered that PEG-200 can control product molecular weight as well as cell productivity for both microorganisms. However, PEG-200 had no significant effect on the polymer compositions. The introduction of PEG-200 in the second-stage culture for 24 hr did not result in any notable cellular toxicity-measured in terms of colony forming unit/mL. Increasing the PEG-200 concentration to 8%, however, caused a decrease in the total polymer yield, the cell productivity as the well as M_n values. Gas chromatography analysis of products indicated that the major repeat units were β-hydroxycaproate, β-hydroxyoctanoate, and β-hydroxydeca noate. Thus, this work initiates new research opportunities for the control of product molecular weight for in-vivo microbial polymer synthetic reactions.     

Optimal culture condition for the production of phenyalanine ammonia lyase from <Emphasis Type="Italic">E. coli</Emphasis>

The effects of culturing conditions on phenylalanine ammonia lyase production by a recombinant E. coli strain were investigated by using a controlled fed-batch fermentation system. In a 5 L fermentor, the optimal composition of the batch medium was 2% glucose, 1% yeast extract, 0.7% K₂HPO₄, 0.8% KH₂PO₄, 0.5% (NH₄)₂SO₄, 0.1% MgSO₄·7H₂O. The optimal feed glucose solution was 50%. Glucose concentration and pH of the culture broth were maintained at about 2.0 g/L and 7.0 during the fed-batch phase, respectively. Following 24-h cultivation, 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) was added and temperature was shifted from 37°C to 42°C to induce pal gene expression. Under optimal conditions, a high productivity of 300 U/g could be achieved after 48 h culture, and a cell density of OD₆₀₀ about 82 was obtained at 52 h culture at 500 r/m stirrer speed and 1 vvm, respectively.     

Polyhydroxybutyrate production accompanied by the effective reduction of chemical oxygen demand (COD) and biological oxygen demand (BOD) from industrial effluent

Industrial effluents are major pollution-causing agents for our environment. Our study focuses on utilizing effluents from different industries for efficient production of Polyhydroxybutyrate (PHB). Presence of PHB was identified by Sudan Black staining method. The PHB production parameters for Pseudomonas aeruginosa MTCC 4673 were studied critically, and it was found that glucose with 8.5 mg/L (0.0550 g PHB/g substrate) PHB concentration yielded the highest among the carbon sources used. Peptone with 8.9 mg/L (0.0524 g PHB/g substrate) of PHB concentration, an incubation period of 48 h and at a pH of 7 yielded the optimum results. These studies were compared with those of Alcaligens latus MTCC 2311. Dairy effluents (DE) and tannery effluents (TE) were considered for the best possible substrate, for the production of PHB in an optimized media. The results indicated that the dairy effluents gave a higher yield of PHB. Amongst various dilution levels studied from 10–100% (v/v), 50% (v/v) concentration of the dairy effluent showed maximum PHB productivity of 0.0582 g PHB/g substrate. A comparison of the chemical oxygen demand (COD) and biological oxygen demand (BOD) from the results, showed a significant removal percentage of 78.97% BOD and 53.482% COD, which highlighted the importance of utilizing effluents for PHB production, in order to reduce the risk of toxic effluent discharge. FT-IR analysis was carried out to confirm the presence of PHB.     

Optimization of medium components for D-ribose production by transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> NJT-1507

Statistical experimental designs were used to optimize the composition of culture media for the production of D-ribose by Bacillus subtilis. A fractional factorial design 2^(5-2) was used to determine medium components that significantly affected D-ribose production. The concentrations of glucose and (NH₄)₂SO₄ were the significant factors. Central composite design and response surface methodology were then used to estimate the quadratic response surface and determine the factor levels for maximum production of D-ribose. Finally, the optimal medium composition was obtained (g/L): glucose, 172.75; (NH₄)₂SO₄, 13.2; yeast powder, 4; corn steep liquor, 8 and MnSO₄, 0.5. This optimization strategy increased D-ribose production from 73.21 g/L to 88.57 g/L, an increase of 22% compared with the original conditions. The D-ribose production yield to glucose concentration was also enhanced from 0.37 g/g to 0.52 g/g. Confirmatory experiments were also performed to demonstrate the accuracy of the model. Under the optimal medium using ammonia to control pH in a 5 L fermenter, the D-ribose yield was increased to 95.28 g/L after 3 days of cultivation at 37 °C.     

Influence of process variables on biooxidation of ferrous sulfate by an indigenous Acidithiobacillus ferrooxidans. Part I: Flask experiments

Biological oxidation of ferrous sulfate by Acidithiobacillus ferrooxidans has proved to be a significant step in the bioleaching of sulfide minerals and the treatment of acid mine drainage. The same bioreaction also has beneficial applications in the desulphurization of coal and removal of hydrogen sulfide from gaseous effluents. In this research, the effects of some process variables such as pH, temperature, elemental sulfur, amount of initial ferrous and magnesium ions on oxidation of ferrous sulfate by a native A. ferrooxidans, which was isolated from a chalcopyrite concentrate, were investigated. All experiments carried out in shake flasks at 33 °C that was obtained as optimum temperature for the specific bacterial growth rate. The optimum range of pH for the maximum growth of the cells and effective biooxidation of ferrous sulfate varied from 2 to 2.3. The maximum biooxidation rate was achieved 1.2 g/L h in a culture initially containing 20.2 g/L Fe²⁺. Mg²⁺ from 20 mg/L to 120 mg/L did not have any effect on the efficiency of the process, while the presence of elemental sulfur had negative effect on the biooxidation.     

Synthesis and properties of silk sericin-<Emphasis Type="Italic">g</Emphasis>-poly(acrylic acid-co-acrylamide) superabsorbent hydrogel

A novel superabsorbent hydrogel has been synthesized with the crosslinking graft copolymerization of acrylic acid (AA) and acrylamide onto the chain of silk sericin. Potassium persulfate (KPS)–sodium sulfite (NaHSO₃) as redox initiation system and N,N′-methylenebisacrylamide (MBA) as crosslinker were used. The structure of the product characterized by Fourier transform infrared absorption spectroscopy and the surface morphology of the hydrogel were observed by scanning electron microscopy. The certain parameters of the graft copolymerization including the monomer, the initiator, the crosslinker concentration, neutralization degree of AA, reaction temperature, and time were systematically optimized to achieve a hydrogel with maximum swelling capacity (2150 g/g). The optimal conditions were initiator 8 mmol/L, MBA 2.5 mmol/L, neutralization degree of AA 75%, reaction temperature 55 °C, and time 6 h. The swelling ratio in salt solutions was also determined (in 0.9% NaCl aqueous solution: 98 g/g). In addition, the swelling capability of the hydrogel was measured in solutions with pH ranged from 1 to 13. The synthesized hydrogel exhibited a pH-dependent character. Water absorbency of the product in aqueous chloride salt solutions has the Na⁺ > Ca²⁺ > Mg²⁺ > Al³⁺ order in the investigated concentration.     

Optimization of medium for phenylalanine ammonia lyase production in <Emphasis Type="Italic">E. coli</Emphasis> using response surface methodology

A culture medium for phenylalanine ammonia lyase (PAL) production in E. coli was developed following preliminary studies by means of response surface methodology (RSM). The medium components having significant effect on the production were first identified by using a fractional factorial design. Then, central composite design (CCD) was used to optimize the medium constituents and explain the combined effects of four medium constituents: glucose, yeast extract, (NH₄)₂HPO₄ and MgSO₄. A quadratic model was found to fit the PAL production. CCD revealed that the optimum values of the test variables for PAL production were glucose 28.2 g/L, yeast extract 5.01 g/L, (NH₄)₂HPO₄ 7.02 g/L and MgSO₄ 1.5 g/L. PAL production of 62.85 U/g, which was in agreement with the prediction, was observed in the verification experiment. In comparison to the production of basal medium, 1.8-fold increase was obtained.     

Simultaneous biofiltration of H<Subscript>2</Subscript>S,NH<Subscript>3</Subscript> and toluene using cork as a packing material

Simultaneous removal of ternary gases of NH₃, H₂S and toluene in a contaminated air stream was investigated over 185 days in a biofilter packed with cork as microbial support. Multi-microorganisms including Nitrosomonas and Nitrobactor for nitrogen removal, Thiobacillus thioparus (ATCC 23645) for H2S removal and Pseudomonas aeruginosa (ATCC 15692), Pseudomonas putida (ATCC 17484) and Pseudomonas putida (ATCC 23973) for toluene removal were used simultaneously. The empty bed residence time (EBRT) was 40–120 seconds and the inlet feed concentration was 50-180 ppmv for NH₃, 30–160 ppmv for H₂S and 40–130 ppmv for toluene, respectively. The observed removal efficiency was 45–100% for NH₃, 96–100% for H₂S, and 10–99% for toluene, respectively. Maximum elimination capacity was 5.5 g/m³/hr for NH₃, >20.4 g/m³/hr for H₂S and 4.5 g/m³/hr for toluene, respectively. During long-term operation, the removal efficiency of toluene gradually decreased, mainly due to depositions of elemental sulfur and ammonium sulfate on the cork surface. The results of microbial analysis showed that nearly the same population density was observed on the surfaces of cork chips collected at each sampling point. Kinetic model analyses showed that there were no particular evidences of interactions or inhibitions among the microorganisms.