The effect of glucose and maltose concentrations on pyruvate and ascorbate production,antioxidant enzyme activities and LPO levels in Fusarium equiseti

Changes in pyruvate and ascorbate production and antioxidant enzyme activities together with lipid peroxidation levels in Fusarium equiseti were investigated in relation to changes in the concentrations of glucose and maltose as carbon sources in the range of 5–25 g/l in Armstrong Fusarium Medium (AFM). The highest pyruvate concentration obtained at 20 g/l maltose was 67.5 ± 0.69 μg/ml while ascorbic acid reached a maximum value at 25 g/l glucose of 1866±26.1 μg/ml The maximum superoxide dismutas (SOD) activities related to increased pyruvate production were determined in AFM medium containing 20 g/l glucose as 41.49±0.65 and maltose as 61.12±0.8 IU/mg. Catalase (CAT) activity variations showed coherence with SOD activity in a medium containing maltose and reached 219.11±2.8 IU/mg while they were decreased with increasing glucose concentration. glutathione peroxidase (GSH-Px) activities in F. equiseti did not change significantly with glucose and maltose concentration and were determined to be 1.21±0.22 and 1.67±0.15 IU/mg, respectively. Minimum lipid peroxidation levels for each carbon source were determined in both 20 g/l maltose and glucose concentrations as 0.9 and 1.62 nmol MDA/g wet weight.     

Crucial factors affecting the nitrile hydratase production of Mesorhizobium sp. F28

Mesorhizobium sp. F28 contains cobalt-NHase, which effectively converts acrylonitrile into acrylamide. When urea was added to the culture medium, the NHase activity was 62.3 U ml^?1 (R2A–R2A/urea) after 22.5 h of cultivation, which was similar to that in the medium without addition (R2A–R2A, 70.0 U ml^?1). The relative activity of the purified NHase was 100%, 92%, 94%, and 92% in the medium containing, respectively, 0 mM, 2 mM, 5 mM, and 10 mM of urea. Urea had no significant effect on the purified NHase activity of Mesorhizobium sp. F28. This research did not observe the NHase production by Mesorhizobium sp. F28 when acrylonitrile was supplemented in the culture medium except that cobalt ions existed. The highest enzyme activity was 328.5 U ml^?1 as cobalt ions were added in the pre-culture and culture medium after 22.5 h of cultivation (R2A/Co-R2A/Co); compared to media without cobalt ions (R2A–R2A, 22.5 h, 70.5 U ml^?1) this is an almost five-fold enhancement. It can be concluded that culture media containing cobalt ions was beneficial for the formation of active NHase of Mesorhizobium sp. F28.     

Metabolic engineering of Torulopsis glabrata for improved pyruvate production

During pyruvate production, ethanol is produced as a by-product, which both decreases the amount of pyruvate and makes the recovery of pyruvate more difficult. Pyruvate decarboxylase (PDC, EC 4.1.1.1), which degrades pyruvate to acetaldehyde and ultimately to ethanol, is a key enzyme in the pyruvate metabolism of yeast. Therefore, to order to increase the yield of pyruvate in Torulopsis glabrata, targeted PDC-disrupted strains were metabolically engineered. First, T. glabrata ura3 strains that were suitable for genetic transformation were isolated and identified through ethyl methansulfonate mutagenesis, 5-fluoroortic acid media selection, and Sacchramyces cerevisiae URA3 complement. Next, the PDC gene in T. glabrata was specifically disrupted through homologous recombinant with the S. cerevisiae URA3 gene as the selective marker. The PDC activity of the disruptants was about 33% that of the parent strain. Targeted PDC gene disruption in T. glabrata was also confirmed by PCR amplification and sequencing of the PDC gene and its mutants, PDC activity staining, and PDC Western blot. The disruptants displayed higher pyruvate accumulation and less ethanol production. Under basal fermentation conditions (see Section 2), the disruptants accumulated about 20 g/L of pyruvate with 4.6 g/L of ethanol, whereas the parental strain (T. glabrata IFO005) only accumulated 7–8 g/L of pyruvate with 7.4 g/L of ethanol. Under favorable conditions in jar fermentation, the disruptants accumulated 82.2 g/L of pyruvate in 52 h.     

Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae

Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142 g/L with production yield of 0.89 g/g and productivity of 3.55 g L⁻¹ h⁻¹ under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast.     

Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains

Saccharomyces cerevisiae has increasingly been engineered as a cell factory for efficient and economic production of fuels and chemicals from renewable resources. Notably, a wide variety of industrially important products are derived from the same precursor metabolite, acetyl-CoA. However, the limited supply of acetyl-CoA in the cytosol, where biosynthesis generally happens, often leads to low titer and yield of the desired products in yeast. In the present work, combined strategies of disrupting competing pathways and introducing heterologous biosynthetic pathways were carried out to increase acetyl-CoA levels by using the CoA-dependent n-butanol production as a reporter. By inactivating ADH1 and ADH4 for ethanol formation and GPD1 and GPD2 for glycerol production, the glycolytic flux was redirected towards acetyl-CoA, resulting in 4-fold improvement in n-butanol production. Subsequent introduction of heterologous acetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase (PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, further increased n-butanol production. Recombinant PDHs localized in the cytosol (cytoPDHs) were found to be the most efficient, which increased n-butanol production by additional 3 fold. In total, n-butanol titer and acetyl-CoA concentration were increased more than 12 fold and 3 fold, respectively. By combining the most effective and complementary acetyl-CoA pathways, more than 100 mg/L n-butanol could be produced using high cell density fermentation, which represents the highest titer ever reported in yeast using the clostridial CoA-dependent pathway.     

Cyanobacterial biomass as N-supplement to agro-waste for hyper-production of laccase from Pleurotus ostreatus in solid state fermentation

The potential application of dry biomass of a cyanobacterium Anacystis nidulans as a supplement in SSF for the production of laccase from Pleurotus ostreatus was evaluated. Experiments were carried out in solid culture using groundnut shell as a basic substrate supplemented with four independent nitrogen sources (ammonium sulphate, urea, yeast extract and dry powder of cyanobacteria). All the four supplements enhanced the enzyme yield, and yeast extract showed precedence over inorganic nitrogenous sources. However, when dry biomass of A. nidulans was used as an additive to groundnut shell (agricultural residues), it supported maximum cell growth (56.83 ± 5.56 mg/g dry substrate) and laccase production (49.21 ± 4.89 U/g dry substrate). Addition of 1 mM copper salt in the medium containing groundnut shell supplemented with yeast extract gave laccase activity of 32.64 ± 3.4 U/g dry substrate. When dry powder of cyanobacterial biomass was used as N-supplement, laccase production enhanced to 65.42 ± 6.48 U/g dry substrate. In addition to the enhancement to enzyme production inhibitory effects of high concentrations of copper was also diminished in the medium having dry cyanobacterial biomass. This study, forms the first report on the potential application of cyanobacterial biomass as an additive for production of laccase by Pleurotus ostraetus MTCC 1804 in solid state fermentation and has relevance in scale-up production of this fungal enzyme of commercial significance.     

Characterization of the extracellular biodemulsifier of Bacillus mojavensis XH1 and the enhancement of demulsifying efficiency by optimization of the production medium composition

The demulsifying bacterium XH1 was identified as a Bacillus mojavensis by the 16S rDNA gene. The extracellular biodemulsifier produced by this species was purified by ethanol extraction and column chromatography through a sephadex and silicon gel column. Preliminary investigation using UV–vis and TLC indicated that the biodemulsifier had two components a protein and a lipopeptide. All major components of the medium, including the sources of soluble and insoluble carbon, nitrogen, phosphate, and metal ions were investigated to improve the biosynthesis and efficiency of the biodemulsifier. The optimal carbon sources were glucose and liquid paraffin. Glucose participated in the biosynthesis of the demulsifier, while liquid paraffin promoted the lipophilicity and secretion of biosurfactants. The absence of yeast extract, ammonium chloride or phosphate (K₂HPO₄/KH₂PO₄) had a negative effect on the production of the biodemulsifier and significantly inhibited its activity. To further enhance the biodemulsifier efficiency, the optimal medium composition was determined using the response surface methodology (RSM) based on the central composite rotation design (CCRD). Using the optimized biodemulsifier production medium: 8.5 g/l glucose; 3% (v/v) liquid paraffin; 1.5 g/l yeast extract; 3.36 g/l NH₄Cl and15 g/l phosphate, the demulsifying ratio increased 35.5% and biodemulsifier yield increased to 2.07 g/l.     

An engineering Escherichia coli mutant with high succinic acid production in the defined medium obtained by the atmospheric and room temperature plasma

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose in nutrient-rich or minimal media anaerobically. Co-expression of heterologous pyruvate carboxylase and nicotinic acid phosphoribosyltransferase in BA002 resulted in a significant increase in cell mass and succinic acid production. Nevertheless, the resultant strain, BA016, still could not grow in a defined medium without tryptone and yeast extract. To solve the problem, a novel atmospheric and room temperature plasma mutation method was employed to generate mutants which can grow in the defined medium. A mutant designated as LL016 was observed to be the best strain that regained the capacity of cell growth and glucose utilization in a defined medium anaerobically. After 120 h of fermentation in the defined medium, 35.0 g/L of glucose was consumed to generate 25.2 g/L of succinic acid. Furthermore, with the highest glucose consumption rate in the dual-phase fermentation, the yield of succinic acid in LL016 reached 0.87 g/g, which was higher than that observed in other strains. From an industrial standpoint, the defined medium is much cheaper than LB medium, which shows a great potential usage for the economical production of succinic acid by LL016.     

Metabolic engineering of Torulopsis glabrata for malate production

The yeast Torulopsis glabrata CCTCC M202019, which is used for industrial pyruvate production, was chosen to explore the suitability of engineering this multi-vitamin auxotrophic yeast for increased malate production. Various metabolic engineering strategies were used to manipulate carbon flux from pyruvate to malate: (i) overexpression of pyruvate carboxylase and malate dehydrogenase; (ii) identification of the bottleneck in malate production by model iNX804; (iii) simultaneous overexpression of genes RoPYC, RoMDH and SpMAE1. Using these strategies, 8.5 g L^–1 malate was accumulated in the engineered strain T.G-PMS, which was about 10-fold greater than that of the control strain T.G-26. The results presented here suggest that T. glabrata CCTCC M202019 is a promising candidate for industrial malate production.     

Enhanced ethanol and chitosan production from wheat straw by Mucor indicus with minimal nutrient consumption

Recently, Mucor indicus was introduced as a promising ethanol producing microorganism for fermentation of lignocellulosic hydrolysates, showing a number of advantages over Saccharomyces cerevisiae. However, high nutrient requirement is the main drawback of the fungus in efficient ethanol production from lignocelluloses. In this study, application of fungal extract as a potential nutrient source replacing all required nutrients in fermentation of wheat straw by M. indicus was investigated. Wheat straw was pretreated with N-methylmorpholine-N-oxide (NMMO) at 120 °C for 1–5 h prior to enzymatic hydrolysis. Hydrolysis yield was improved at least by 6-fold for 3 h pretreated straw compared with that of untreated one. A fungal extract was produced by autolysis of M. indicus biomass, an unavoidable byproduct of fermentation. Maximum free amino nitrogen (2.04 g/L), phosphorus (1.50 g/L), and total nitrogen (4.47 g/L) as well as potassium, magnesium, and calcium in the fungal extract were obtained by autolysis of the biomass at 50 °C and pH 5.0. The fungal extract as a nutrient-rich supplement substituted yeast extract and all other required minerals in fermentation and enhanced the ethanol yield up to 92.1% of the theoretical yield. Besides, appreciate amounts of chitosan were produced as another valuable product of the autolysis.     

Application of response surface methodology for glucan production from Leuconostoc dextranicum and its structural characterization

Sequential optimization strategy based on statistical experimental designs was employed to enhance glucan production by Leuconostoc dextranicum NRRL B-1146 in flask culture. A two-level Plackett–Burman design was employed first where 11 variables were studied for their influence on glucan production. Sucrose, peptone and yeast extract were the most significant variables improving glucan production. A three-level Box–Behnken factorial design was employed for maximizing the glucan production. A mathematical model was developed to show the effects of each medium component and their combinatorial interactions on glucan production. The optimal medium composition for maximum glucan production was sucrose 5.95%, peptone 0.52% and yeast extract 2.9%. This composition predicted 1063 mg/l glucan, the experimentally found glucan was 1015 ± 4.5 mg/l that showed a good agreement with the predicted value. The purified glucan was homogenous and its structural characteristics investigated by FT-IR, ¹H NMR and ¹³C NMR spectroscopic techniques showed that it contained α-(1 → 6) and α-(1 → 4) linkages.     

Newly developed medium and strategy for bacterial cellulose production

Bacterial cellulose (BC) has unique properties, such as high crystallinity, a high degree of polymerisation, high tensile strength and high purity, compared with native cellulose. In this study, a previously determined BC production medium was improved in static culture, and the production cost was evaluated and compared with molasses and with other defined media, such as Hestrin–Schramm, Zhou, Yamanaka and Park, using Gluconacetobacter xylinus. In addition to this analysis, because the surface area/volume ratio is an important parameter in static culture, different surface area/volume ratios were analysed in the range of 0.2–1.46. The defined medium (M1A05P5) and culture type contained glucose (10 g/L), yeast extract (10 g/L), peptone (7 g/L), acetic acid (1.5 ml/L), and ethanol (5 ml/L), and the pH was adjusted to 5.0 in static culture. The highest productivity was observed in the M1A05P5 medium that was 5-fold higher than either molasses or Park's medium. Although the molasses medium was proposed as a cost-effective medium, the production price of BC was the lowest in the M1A05P5 medium. Therefore, the newly developed medium and strategy were highly promising candidates for the industrial-scale production of BC.     

A new semi-selective medium for Fusarium graminearum,F. proliferatum,F. subglutinans and F. verticillioides in maize seed

Zea mays L., known also as corn and maize, is the most important crop according to the amount of tonnes produced each year. Fungi cause significant destruction of maize in the field as well as during storage rendering the grain unsuitable for human consumption by decreasing its nutritional value and by producing mycotoxins that are detrimental to both human and animal health. Fusarium species are widely distributed and are amongst the most frequently isolated fungal species by plant pathologists. Due to the fact that the Fusarium species involved in maize ear rot vary in fungicide sensitivity, pathogenicity as well as in their capability to produce mycotoxins, accurate quantification and identification is of paramount significance. Currently no method has been developed to test for Fusarium species in maize seed that has been validated and published by the International Seed Testing Association (ISTA). Malachite green agar 2.5 ppm (MGA 2.5) is a potent selective medium for isolation and enumeration of Fusarium spp. In this study, eight different media compositions, potato dextrose agar (PDA), PDA + malachite green oxalate, corn meal agar, 1/2 PDA + malachite green oxalate, 1% malt agar, carnation leaf agar supplemented with potassium chloride (KCLA), malachite green agar (MGA 2.5) and MGA 2.5 + sterile carnation leaf pieces were compared using four Fusarium species (F. graminearum, F. proliferatum, F. subglutinans and F. verticillioides) and five commonly encountered saprophytic fungi (Aspergillus niger, Penicillium crustosum, P. digitatum, Trichoderma harzianum and Rhizopus stolonifer). The maize kernels were surface disinfected using three concentrations of sodium hypochlorite (0.5%, 1% and 1.5% NaOCl) and for different time intervals (1 min, 3 min, 5 min and 10 min). The effect of black-blue light (365 nm) on sporulation of the fungi was also investigated. Surface disinfection of maize seeds with 1% NaOCl for 5 min provided consistent results. PDA, 1/2 PDA, 1% malt agar and KCLA allowed profuse growth of the Fusarium species as well as saprophytes. Media that contained malachite green oxalate was most inhibitory to the radial colony growth of the saprophytes and the Fusarium species. The Fusarium species growing on these media formed underdeveloped morphological structures, thereby obscuring accurate identification. MGA 2.5 showed better hindering of the saprophytes in some instances. MGA 2.5 amended with sterile carnation leaf pieces was the most satisfactory medium in hindering the growth of the saprophytes while allowing adequate sporulation by the four Fusarium species to permit accurate identification. The media also resulted in higher F. verticillioides and lower saprophytic fungal isolation frequency when compared to the other media tested.     

Optimization of culture medium for cordycepin production using Cordyceps militaris mutant obtained by ion beam irradiation

The effect of medium components on cordycepin production by Cordyceps militaris mutant obtained by ion beam irradiation was investigated. According to the response surface analysis using a central composite design for the prospective mutant G81-3, the predicted optimal concentrations of glucose as the carbon source and the yeast extract as the nitrogen source were 86.2 g/l and 93.8 g/l, respectively, and 6.84 g/l cordycepin was obtained. To date, this is the highest value for cordycepin production. The optimal concentrations of glucose and yeast extract for cordycepin production of the mutant was much higher than that of control (wild strain) and the cordycepin production was 2.79 times higher. Therefore, this new mutant will be a promising strain for future higher cordycepin production at industrial levels.     

Evaluation of single cell oil from Aureobasidium pullulans var. melanogenum P10 isolated from mangrove ecosystems for biodiesel production

In this study, the yeast strain P10 which was identified to be a member of Aureobasidium pullulans var. melanogenum isolated from the mangrove ecosystems was found to be able to accumulate high content of oil in its cells. After optimization of the medium for lipid production and cell growth by the yeast strain P10, it was found that 8.0 g of glucose per 100 ml, 0.02 g of yeast extract per 100 ml, 0.02 g of ammonium sulfate per 100 ml, pH 6.0 in the medium were the most suitable for lipid production. During 10-l fermentation, a titer was 66.3 g oil per 100 g of cell dry weight, cell mass was 1.3 g per 100 ml, a yield was 0.11 g of oil per g of consumed sugar and a productivity was 0.0009 g of oil per g of consumed sugar per h within 120 h. At the same time, only 0.07 g of reducing sugar per 100 ml was left in the fermented medium. The compositions of the fatty acids produced were C_16:0 (26.7%), C_16:1(1.7%), C_18:0 (6.1%), C_18:1 (44.5%), and C_18:2 (21.0%). The biodiesel produced from the extracted lipid could be burnt well.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor

Lactobacillus kefiranofaciens is non-pathogenic gram positive bacteria isolated from kefir grains and able to produce extracellular exopolysaccharides named kefiran. This polysaccharide contains approximately equal amounts of glucose and galactose. Kefiran has wide applications in pharmaceutical industries. Therefore, an approach has been extensively studied to increase kefiran production for pharmaceutical application in industrial scale. The present work aims to maximize kefiran production through the optimization of medium composition and production in semi industrial scale bioreactor. The composition of the optimal medium for kefiran production contained sucrose, yeast extract and K₂HPO₄ at 20.0, 6.0, 0.25 g L⁻¹, respectively. The optimized medium significantly increased both cell growth and kefiran production by about 170.56% and 58.02%, respectively, in comparison with the unoptimized medium. Furthermore, the kinetics of cell growth and kefiran production in batch culture of L. kefiranofaciens was investigated under un-controlled pH conditions in 16-L scale bioreactor. The maximal cell mass in bioreactor culture reached 2.76 g L⁻¹ concomitant with kefiran production of 1.91 g L⁻¹.     

Production and characterization of curdlan by Agrobacterium sp.

Agrobacterium sp. was studied for the production of curdlan by conventional one-factor-at-a-time technique and response surface methodology. Factors such as initial pH, urea concentration, sucrose concentration having the greatest influence on the curdlan production were identified. By using response surface methodology (RSM), the curdlan production by Agrobacterium sp. was increased significantly by 109%, from 2.4 g/L to 5.02 g/L when the strain was cultivated in the optimal medium developed by RSM as compared to conventional one-factor-at-a-time technique. The curdlan production rate of 0.84 g/(L h) was obtained when Agrobacterium sp. was cultivated in the optimal medium developed by RSM, which was the highest curdlan production rate reported to date. The infrared (IR) and NMR spectra, the thermogram of DSC and pattern of X-ray diffraction for the curdlan of the present study were almost identical to those of the authentic curdlan sample (from Alcaligenes faecalis; Sigma). The purified curdlan was a linear polysaccharide composed of exclusively β-(1,3)-glucosidic linkages with the molecular weight of 160,000 Da by GPC. The crystalline melting point (T_m), glass transition temperature (T_g) and X-ray diffraction of the sample indicated low crystallinity in the structure.     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.