Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.     

Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4

Microbial fermentation under anaerobic and microaerobic conditions has been used for the production of 1,3-propanediol (1,3-PD), a monomer used to produce polymers such as polytrimethylene terephthalate. In this study, we screened microorganisms using the high throughput screening method and isolated the Klebsiella pneumoniae AJ4 strain, which is able to produce 1,3-PD under aerobic conditions. To obtain the maximum 1,3-PD concentration from glycerol, the response surface methodology based on a central composite design was chosen to show the statistical significance of the effects of glycerol, peptone, and (NH₄)₂SO₄ on 1,3-PD production by K. pneumoniae AJ4. The optimal culture medium factors for achieving maximum concentrations of 1,3-PD included glycerol, 108.5 g/L; peptone, 2.72 g/L; and (NH₄)₂SO_4, 4.38 g/L. Under this optimum condition, the maximum concentration of 1,3-PD, 54.76 g/L, was predicted. A concentration of about 52.59 g/L 1,3-PD was obtained using the optimized medium during 26-h batch fermentation, a finding that agreed well with the predicted value.     

Fast conversion of glycerol to 1,3-propanediol by a new strain of Klebsiella pneumoniae

Five bacterial strains screened from a batch of 39 samples could convert glycerol anaerobically to 1,3-propanediol (1,3-PD). One of the strains, XJ-Li, which could synthesize 1,3-PD with a higher concentration, was identified and characterized. Phylogenetic analysis of the strain XJ-Li included the study of morphology, physiological and biochemical characteristics. In addition, 16SrDNA sequences were created. The results indicated that this strain is a member of Klebsiella pneumoniae. The optimal cultivation parameters for pH and temperature were determined as 8.0 and 40 °C, respectively. The optimized nitrogen source and carbon source were 6.0 g/L of (NH₄)₂SO₄ and 20 g/L of glycerol, respectively. After 8 h in batch fermentation, both the 1,3-PD concentration and glycerol consumption reached the maximum, with 12.2 g/L of 1,3-PD and 1.53 g/L h of productivity, and a molar yield of 1,3-PD to glycerol of 0.75. Fed-batch fermentation also indicated a higher molar yield of 0.70, and the concentration of 1,3-PD reached 38.1 g/L after 66.4 g/L of glycerol consumption. The results of batch and fed-batch fermentations demonstrated that K. pneumoniae XJ-Li would be an excellent 1,3-PD producer.     

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH₄)₂SO₄ and soybean flour as nitrogen sources and KH₂PO₄ at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.     

High-level production of 1,3-propanediol from crude glycerol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> AKR102a

The aim of this study was to optimize a biotechnological process for the production of 1,3-propanediol (1,3-PD) based on low-quality crude glycerol derived from biodiesel production. Clostridium butyricum AKR102a was used in fed-batch fermentations in 1-L and 200-L scale. The newly discovered strain is characterized by rapid growth, high product tolerance, and the ability to use crude glycerol at the lowest purity directly gained from a biodiesel plant side stream. Using pure glycerol, the strain AKR102 reached 93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h). With crude glycerol under the same conditions, 76.2 g/L 1,3-PD was produced with a productivity of 2.3 g/(L*h). These are among the best results published so far for natural producers. The scale up to 200 L was possible. Due to the simpler process design, only 61.5 g/L 1,3-PD could be reached with a productivity of 2.1 g/(L*h).     

Optimizing emulsan production of <Emphasis Type="Italic">A. venetianus</Emphasis> RAG-1 using response surface methodology

Statistical experimental design was used to optimize medium constituents for emulsan production by Acinetobacter venetianus RAG-1 in batch cultivation. The factors affecting emulsan production were screened by a two-level factorial design, and the optimal concentration of medium constituents for emulsan production were determined by the method of steepest path ascent and central composite experimental design. Experimental results showed that the optimal medium constituents were 9.16 g/L ethanol, 8.2 g/L KH₂PO₄, 23.32 g/L K₂HPO₄, 5.77 g/L (NH₄)₂SO₄ and 0.354 g/L MgSO₄•7H₂O. Under this optimal composition, the predicted emulsan production was 72.198 mg/L, and experimental value was 73.312 mg/L for 80 h culture in the shake flasks, and the emulsan yield by A. venetianus RAG-1 was enhanced nearly 1.48-fold (from 49.5 to 73.312 mg/L). Based on the results, we identify the optimal medium constituents for emulsan production and could take advantage of strategy for scale up the fermentation of emulsan production.     

Development of fermentation process for PLA-degrading enzyme production by a new thermophilic Actinomadura sp. T16-1

The fermentation process for a poly (L-lactide) (PLA)-degrading enzyme production by a newly isolate of thermophilic PLA-degrading Actinomadura sp. T16-1 was investigated. The strain produced 33.9 U/mL of enzyme activity after cultivation at 50°C under shaking of 150 rpm for 96 h in a medium consisting of (w/v) 0.05% PLA film, 0.2% gelatin, 0.4% (NH₄)₂SO₄, 0.4% K₂HPO₄, 0.2 % KH₂PO₄, and 0.02% MgSO₄ · 7H₂O. The optimal concentration of PLA film and gelatin obtained by response surface methodology (RSM) for the highest production of PLA-degrading enzyme was 0.035% (w/v) and 0.238% (w/v), respectively. Under these conditions, the model predicted 40.4 U/mL of PLA-degrading activity and the verification of the optimization showed 44.6 U/mL of PLA-degrading enzymatic activity in the flasks experiment. The maximum PLA-degrading activity reached 150 U/mL within 72 h cultivation in the 3-L airlift fermenter.     

Production of 1,3-propanediol from Glycerol by Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Incompatible Plasmids System

1,3-Propanediol (1,3-PD) has numerous applications in polymers, cosmetics, foods, lubricants, and medicines as a bifunctional organic compound. The genes for the production of 1,3-PD in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, and gdrAB, which encodes glycerol dehydratase reactivating factor, are naturally under the control of different promoters and are transcribed in different directions. These genes were coexpressed in E. coli using two incompatible plasmids (pET28a and pET22b) in the presence of selective pressure. The recombinant E. coli coexpressed the glycerol dehydratase, 1,3-propanediol oxidoreductase and reactivating factor for the glycerol dehydratase at high levels. In a fed-batch fermentation of glycerol and glucose, the recombinant E. coli containing these two incompatible plasmids consumed 14.3 g/l glycerol and produced 8.6 g/l 1,3-propanediol. In the substitution case of yqhD (encoding alcohol dehydrogenase from E. coli) for dhaT, the final 1,3-propanediol concentration of the recombinant E. coli could reach 13.2 g/l.     

Impact of different pH control agents on biopesticidal activity of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> during the fermentation of starch industry wastewater

Different pH control agents (NaOH/H₂SO₄—SodSulp, NaOH/CH₃COOH—SodAcet, NH₄OH/CH₃COOH—AmmoAcet and NH₄OH/H₂SO₄—AmmoSulp) were used to investigate their effects on growth, enzyme production (alkaline protease and amylase), and entomotoxicity of Bacillus thuringiensis var. kurstaki HD-1 (Btk) against eastern spruce budworm larvae (Choristoneura fumiferana) using starch industry wastewater (SIW) as a raw material in a 15-l fermentor. AmmoSulp and SodSulp were found to be the best pH control agents for alkaline protease and amylase production, respectively; whereas, the fermented broth obtained by using SodAcet as pH control agents recorded the highest delta-endotoxin production of 1043.0 mg/l and entomotoxicity value 18.4 × 10⁹ SBU/l. Entomotoxicity of re-suspended centrifuged pellet in one-tenth of original volume in case of SodAcet as pH control agents was 26.7 × 10⁹ SBU/l and was the highest value compared to three other pH control agents.     

Enhancing yield of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by controlling NH<Subscript>4</Subscript><Superscript>+</Superscript> concentration

Yield of S-adenosylmethionine was improved significantly in recombinant Pichia pastoris by controlling NH₄ ⁺ concentration. The highest production rate was 0.248 g/L h when NH₄ ⁺ concentration was 450 mmol/L and no repression of cell growth was observed. Within very short induction time (47 h), 11.63 g/L SAM was obtained in a 3.7 L bioreactor.     

Characterization of 1,3-propanediol oxidoreductase (DhaT) from <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> J2B

1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (k _cat , 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (K _m , mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD⁺) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag⁺ and Hg²⁺. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.     

Microbial production of 1,3-propanediol from glycerol by Klebsiella pneumoniae under micro-aerobic conditions up to a pilot scale

1,3-Propanediol (1,3-PD) can be produced from glycerol by Klebsiella pneumoniae under micro-aerobic conditions. Recently, this fed-batch fermentation process has been successfully scaled up to 1 m³. The final 1,3-PD concentration, molar yield and volumetric productivity of 72 g l⁻¹, 57% and 2.1 g l⁻¹ h⁻¹, respectively, are close to those of 75 g l⁻¹, 61%, and 2.2 g l⁻¹ h⁻¹ under anaerobic conditions. This process would be suitable for the production of 1,3-PD on a large scale.     

Development of a novel solid-state fermentation strategy for the production of poly-3-hydroxybutyrate using polyurethane foams by Bacillus sphaericus NII 0838

The extensive use of synthetic plastics has caused serious waste disposal problems in our environment. Poly-3-hydroxybutyrates (PHB) are eco-friendly bacterial polyesters which are produced under unbalanced nutrient conditions. Few reports are available on PHB production by solid state fermentation (SSF). We have developed a novel SSF bioprocess in which polyurethane foam (PUF) is used as a physical inert support for the production of PHB by Bacillus sphaericus NII 0838. Media engineering for optimal PHB production was carried out using response surface methodology (RSM) adopting a Box–Behnken design. The factors optimized by RSM were inoculum size, pH and (NH₄)₂SO₄ concentration. Under optimized conditions—6.5 % inoculum size, 1.7 % (w/v) (NH₄)₂SO₄ and pH 9.0—PHB production and biomass were 0.169?±?0.03 and 0.4?±?0.002 g/g PUF, respectively. This is the first report on PHB production by SSF using PUF as an inert support. Our results demonstrate that SSF can be used as an alternative strategy for the production of PHB.     

Polysaccharide production by Aureobasidium pullulans: factors affecting polysaccharide formation

Aureobasidium pullulans NRRL 6220 synthesized polysaccharide most actively in media containing sucrose, fructose or maltose with (NH₄)₂SO₄ (0.6 g/l) or ammonium acetate giving greatest yields of the polysaccharide. With (NH₄)₂SO₄ at 1.2 g/l, production of polysaccharide was decreased considerably. Polysaccharide production was highest with an initial pH of 6.5 while biomass formation was better below an initial pH of 5.5. Optimum phosphate concentration for polysaccharide production was 0.03 m.S.M. Badr-Eldin, H.G. El-Masry and O.A. Abd El-Rahman are with the Microbial Chemistry Department, National Research Center, Dokki, Cairo, Egypt; F.H.A. Mohamad is with the Chemical Engineering and Pilot Plant Department, National Research Center, Dokki, Cairo, Egypt. O.M. El-Tayeb is with the Microbiology Department, Faculty of Pharmacy, Cairo University, Egypt.     

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH₄)₂SO₄, KH₂PO₄, or Mg²⁺ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.     

Salt‐enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in Actinomadura namibiensis

Salt‐enhanced cultivation as a morphology engineering tool for the filamentous actinomycete Actinomadura namibiensis was evaluated in 500‐mL shaking flasks (working volume 100 mL) with the aim of increasing the concentration of the pharmaceutically interesting peptide labyrinthopeptin A1. Among the inorganic salts added to a complex production medium, the addition of (NH₄)₂SO₄ led to the highest amount of labyrinthopeptin A1 production. By using 50 mM (NH₄)₂SO₄, the labyrinthopeptin A1 concentration increased up to sevenfold compared to the non‐supplemented control, resulting in 325 mg L^?1 labyrinthopeptin A1 after 10 days of cultivation. The performance of other ammonium‐ and sulfate‐containing salts (e.g., NH₄Cl, K₂SO₄) was much lower than the performance of (NH₄)₂SO₄. A positive correlation between the uptake of glycerol as one of the main carbon sources and nongrowth‐associated labyrinthopeptin productivity was found. The change in the cell morphology of A. namibiensis in conjunction with increased osmolality by the addition of 50 mM (NH₄)₂SO₄, was quantified by image analysis. A. namibiensis always developed a heterogeneous morphology with pellets and loose mycelia present simultaneously. In contrast to the non‐supplemented control, the morphology of (NH₄)₂SO₄‐supplemented cultures was characterized by smaller and circular pellets that were more stable against disintegration in the stationary production phase.     

Microbial production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations

1,3-Propanediol (1,3-PD) was produced by Klebsiella pneumoniae using crude glycerol obtained from biodiesel production. The 1,3-PD concentration of 51.3 g/l⁻¹ on crude glycerol from alkali-catalyzed methanolysis of soybean oil was comparable to that of 53 g/l⁻¹ on crude glycerol derived from a lipase-catalyzed process. The productivities of 1.7 g l⁻¹ h⁻¹ on crude glycerol were comparable to that of 2 g l⁻¹ h⁻¹ on pure glycerol. It could be concluded that the crude glycerol could be directly converted to 1,3-PD without any prior purification.     

Xylanase production under solid-state fermentation and its characterization by an isolated strain of <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> in India

Summary A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid state fermentation. Corncobs were found as the best substrates for high yield of xylanases with poor cellulase production. The influence of various parameters such as temperature, pH, moistening agents, moisture level, nitrogen sources and pretreatment of substrates were evaluated with respect to xylanase yield, specific activity and cellulase production. Influence of nitrogen sources on protease secretion was also examined. Maximum xylanase production (3065 U/g) was obtained on untreated corncobs moistened with modified Mandels and Strenberg medium, pH 5.0 at 1 5 moisture levels at 30 °C in 4 days of cultivation. Submerged fermentation under the same conditions gave higher yield (3300 U/g) in 5 days of cultivation, but productivity was less. Ammonium sulphate fractionation yielded 3.56-fold purified xylanase with 76% recovery. Optimum pH and temperature for xylanase activity were found to be 5.3 and 50 °C respectively. Kinetic parameters like K_m and V_max were found to be 3.58 mg/ml and 570 μmol/mg/min. Activity of the enzyme was found to be enhanced by cystiene hydrochloride, CoCl₂, xylose and Tween 80, while significantly inhibited by Hg⁺⁺, Cu⁺⁺ and glucose. The enzyme was found to be stable at 40 °C. The half life at 50 °C was 57.53 min. However thermostability was enhanced by glycerol, trehalose and Ca⁺⁺. The crude enzyme was stable during lyophilization and could be stored at less than 0 °C.     

Statistical optimization of cellulases production by <Emphasis Type="Italic">Penicillium</Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis> QML-2 under solid-state fermentation and primary application to chitosan hydrolysis

Solid-state fermentation conditions for cellulases production by a newly isolated Penicillium chrysogenum QML-2 were investigated using statistical methods. At first, significant variables for cellulases production including (NH₄)₂SO₄, initial pH and inoculum size were screened by using Plackett-Burman Design. Then the optimal regions of the significant variables were investigated by using the method of steepest ascent. Finally, central composite design and response surface analysis were adopted to determine the optimal values of the significant variables and investigate the combined effects of each variable’s pair on cellulases production. The results showed that the optimal ranges of (NH₄)₂SO₄ concentration, initial pH and inoculum size for three types of cellulases activities were 1.97–2.15 g, pH 4.32–4.41 and 13.3–13.7% (v/w), respectively. Using the mixture of corn stover powder and wheat bran (CSP/WB, 1/1) as carbon source, the optimization resulted in 370.15, 101.76 and 321.56 U/g for maximal endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. Compared with maximum values of cellulases activities (endoglucanase activity 85.21 U/g, filter paper activity 16.62 U/g and β-glucosidase activity 67.68 U/g) obtained under unoptimized conditions, the optimization resulted in 3.34, 5.12 and 3.75 folds improvement for endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. For chitosan hydrolysis, the crude cellulases had the optimal temperature of 55°C, pH of 4.4 and exhibited Michaelis constant (K _m) value of 8.34 mg/ml and maximum velocity (V _max) of 2.21 μmol glucosamine/min by 1 ml of the crude cellulases.