Improved production of curdlan with concentrated cells ofAgrobacterium sp.

The addition of a limited concentration of yeast extract to a minimal salt medium (MSM) enhanced cell growth and increased the production of curdlan whereas nitrogenlimitation was found to be essential for the higher production of curdlan byAgrobacterium sp. ATCC 31749. As the amount of the inoculum increased, the cell growth as well as the production of curdlan also increased in the MSM without a nitrogen source. The cell growth and production of curdlan increased as the initial pH of the medium decreased as low as 5.0. The conversion rate and concentration of curdlan from 2% (w/v) glucose in the MSM with concentrated cells under nitrogen deletion was 67% and 13.4 g/L, respectively. The highest conversion rate of curdlan under the conditions optimized in this study was 71% when the glucose concentration was 1% (w/v).     

Corynecin (chloramphenicol analogs) fermentation studies: Selective production of Corynecin I by Corynebacterium hydrocarboclastus grown on acetate

Corynebacterium hydrocarboclastus KY 8835 grew in the acetate medium and accumulated 28mM Corynecins which was the highest production yield among the processes using various carbon sources. Selective production of Corynecin I (over 90% of all Corynecins), which had been desired for increase of the product yield, was achieved in this acetate medium. To keep the concentration of acetate, ammonium, and phosphate ions in the optimum range throughout the fermentation, a solution containing CH₃COOH (50%), CH₃COONH₄ (9%), and KH₂PO₄(0.2%) was fed continuously to the culture medium as the pH controlling agent. The addition of KCl (1%) and NaCl (1%) to the medium at 12 hr after inoculation stimulated the production of Corynecins.     

CITRIC ACID PRODUCTION BY Candida SPECIES GROWN ON A SOY-BASED CRUDE GLYCEROL

Citric acid was produced by five species of the yeast Candida after growth on a medium containing soy biodiesel-based crude glycerol. After growth on a medium containing 10 g L^?1 or 60 g L^?1 crude glycerol for 168 hr at 30°C, Candida parapsilosis ATCC 7330 and C. guilliermondii ATCC 9058 produced the highest citric acid levels. On 10 g L^?1 or 60 g L^?1 crude glycerol for 168 hr at 30°C, the citric acid level produced by C. parapsilosis ATCC 7330 was 1.8 g L^?1 or 11.3 g L^?1, respectively, while C. guilliermondii ATCC 9058 produced citric acid concentrations of 3.0 g L^?1 or 10.4 g L^?1, respectively. Biomass production by C. guilliermondii ATCC 9058 on 10 g L^?1 or 60 g L^?1 crude glycerol for 168 hr at 30°C was highest at 1.2 g L^?1 or 6.9 g L^?1, respectively. The citric acid yields observed for C. guilliermondii ATCC 9058 after growth on 10 g L^?1 or 60 g L^?1 crude glycerol (0.35 g g^?1 or 0.21 g g^?1, respectively) were generally higher than for the other Candida species tested. When similar crude glycerol concentrations were present in the culture medium, citric acid yields observed for some of the Candida species utilized in this study were about the same or higher compared to citric acid yields by Yarrowia lipolytica strains. Based on the findings, it appeared that C. guilliermondii ATCC 9058 was the most effective species utilized, with its citric acid production being similar to what has been observed when citric acid-producing strains of Y. lipolytica were grown on crude glycerol under batch conditions that could be of significance to biobased citric acid production.     

Optimization of uracil addition for curdlan (β-1→3-glucan) production by Agrobacterium sp.

Uracil, acting as a precursor of UDP-glucose, served as an activator on the production of curdlan with Agrobacterium sp. (ATCC 31750). The time of adding uracil was important to improve curdlan production. When uracil was added after ammonium depletion (at 26 h), it was used as a nitrogen source for cell growth. Although the cell concentration increased, the curdlan production was decreased. If uracil was added at 46 h, then uracil was degraded slowly but still activated curdlan production. With the addition of both sucrose (200 g) and uracil (1.5 g), the curdlan production was increased up to 93 g l^–1 after 160 h fermentation.     

Optimal production of curdlan by Agrobacterium sp. with feedback inferential control of optimal pH profile

The pH control was important for curdlan production with Agrobacterium sp. ATCC31750. Specific cell growth rate was the highest at pH 7 and the specific curdlan production rate was at pH 5.5. The pH profiles maximizing curdlan production was changed from pH 7 optimal for cell growth to pH 5.5 optimal for curdlan production after ammonium consumption. The feedback inferential control methods, with easily measurable variables such as NaOH addition for pH control and dissolved oxygen (DO), were also applied. The pH was successfully controlled to follow optimal profiles and the maximal production of curdlan (60 g l^–1 in 120 h) was achieved with feedback optimal control.     

A two stage continuous process for the production of thermogelable curdlan-type exopolysaccharide

Summary A laboratory-scale, two-stage continuous process for the production of curdlan-type exopolysaccharide has been operated in steady-state for 500hr. Two continuous flow, constant volume fermenters are connected in series. A stable, curdlan-producing strain of Alcaligenes faecalis var myxogenes ATCC 31749 is grown aerobically in a nitrogen-limited chemostat operating near D_max at 0.24 hr^–1. The effluent is introduced directly into a second larger constant volume fermenter which is being simultaneously fed a glucose solution at a fixed rate. Under sub-optimal conditions associated with curdlan production, the observed Qp was 0.05 g curdlan/g cell/hr. At a biomass level of 4 g/L in the second stage, curdlan was present at 10 g DW/L and the volumetric productivity was 0.2 g/g cell/hr. The substrate (glucose) conversion efficiency was 42%. The process is described in patents applied for on behalf of George Weston Ltd. (Toronto, Canada).     

Enhanced curdlan production in <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749 by addition of low-polyphosphates

A large amount of adenosine triphosphate with high energy phosphate bonds is required for uridine triphosphate regeneration during curdlan biosynthesis by Agrobacterium sp. ATCC 31749. To supply high energy for curdlan synthesis, three low-polyphosphates (Na₄P₂O₇, Na₅P₃O₁₀, and (NaPO₃)₆) with higher energy phosphate bonds were employed to substitute for KH₂PO₄-K₂HPO₄ in fermentation medium. Two genes encoding the polyphosphate metabolizing enzymes, polyphosphate kinase and exopolyphosphatase, were amplified and showed 95% homology to those in Agrobacterium sp. C58 by sequence analysis. The curdlan yields were enhanced by 23 and 134% when phosphate concentrations 0.024 mol/L of Na₅P₃O₁₀ and 0.048 mol/L of (NaPO₃)₆ respectively, were added in the medium. The maximum curdlan yield of 30 ± 1.02 g/L was obtained with the addition of 0.048 mol/L of (NaPO₃)₆ with 5 g/L CaCO₃ in the medium. When CaCO₃ was removed from the culture and the three lowpolyphosphates were added, the pH and biomass yield dropped remarkably and little or no curdlan was produced. The culture containing 0.048 mol/L of (NaPO₃)₆ was mixed with KH₂PO₄-K₂HPO₄ and CaCO₃ in the medium, but showed no effect on curdlan production. However, curdlan yield was improved by 49 ∼ 60% when CaCO₃ was removed from the medium and KH₂PO₄-K₂HPO₄ acted as a buffer. It appears that the positive effect of (NaPO₃)₆ on curdlan production required the buffering capacity of CaCO₃ and the absence of KH₂PO₄-K₂HPO₄ competing as a phosphate supplier.     

Changes of Curdlan Biosynthesis and Nitrogenous Compounds Utilization Characterized in <Emphasis Type="Italic">ntrC</Emphasis> Mutant of <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749

The regulatory function of global regulator NtrC on curdlan biosynthesis and nitrogen consumption under nitrogen-limited condition in Agrobacterium sp. ATCC 31749 was investigated. The ntrC mutant of Agrobacterium sp. was constructed by homologous recombination. The ability to utilize NH₄Cl and KNO₃ was impaired in the mutant. Other nitrogenous compounds, such as glutamic acid and glutamine, were utilized normally. Curdlan production capability was impaired severely in the mutant. Curdlan production was 5-fold lower than the wild type strain in batch fermentation with NH₄Cl as the sole nitrogen source. However, up to 6.5 g l⁻¹ of a newly found alkali-insoluble biopolymer was produced by the ntrC mutant when glutamic acid was used as nitrogen source. The new biopolymer had glycosidic bond and hydroxyl group but no β-configuration absorption peak on IR spectrum was found as different from curdlan. In addition, the mutant exhibited a rapid morphological change from the dot to rod form. These results deduced that the global regulator NtrC was involved in curdlan and other biopolymer biosynthesis in Agrobacterium sp. ATCC 31749 in response to nitrogen-limited condition.     

Component identification of electron transport chains in curdlan-producing <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis

Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb ₃-type terminal oxidase and cytochrome caa ₃-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.     

NtrC-dependent regulatory network for curdlan biosynthesis in response to nitrogen limitation in Agrobacterium sp. ATCC 31749

The mechanism of nitrogen signal regulating curdlan biosynthesis in Agrobacterium sp. ATCC 31749 was investigated. Under nitrogen limitation, more carbon flux is directed to curdlan synthesis with low specific growth rate. When ntrB and ntrC genes in Agrobacterium sp. were inactivated, NH₄Cl utilization ability was significantly impaired in the ntrB and ntrC mutants and curdlan production was significantly reduced. Through proteomic analysis, nearly 40 proteins did not express in ntrC mutant compared with wild type strain. The levels of 22 proteins were significantly increased and 21 proteins were repressed after nitrogen exhaustion. Phosphoglucomutase activity in Agrobacterium sp. was also decreased. However, phosphoglucomutase activity in the ntrC mutant did not change. On that basis, an NtrC-dependent regulatory network for curdlan biosynthesis in response to nitrogen limitation in Agrobacterium sp. ATCC 31749 is proposed.     

ntrB基因对Agrobacterium sp.ATCC31749合成胞外多糖的调控研究

利用三亲本接合方法将含有ntrB基因两端同源序列的自杀载体pJQ-ntrB-cat导入土壤杆菌(Agrobacterium sp.ATCC 31749)中,获得了ntrB基因突变株.结果表明,ntrB突变株对NH4Cl和KNO3的利用能力有所下降.当分别以谷氨酸和谷氨酰胺为氮源时,ntrB突变株生长状况与野生菌相同.n...     

Xylitol production by Candida species grown on a grass hydrolysate

Xylitol was produced by selected species of the yeast Candida after growth on a medium containing a hydrolysate of the North American perennial prairie grass big bluestem. The grass was hydrolysed by a combination of dilute acid and enzymatic treatments. After growth on the medium for 120 h at 30 °C, Candida tropicalis ATCC 750 produced a 1.4-fold higher level of xylitol than did C. tropicalis ATCC 20215 while biomass production by C. tropicalis ATCC 750 was 1.7-fold higher than Candida guilliermondii ATCC 20216. The xylitol yields observed for C. tropicalis ATCC 750, Candida mogii ATCC 18364 and C. guilliermondii ATCC 20216 were at least 1.4-fold higher than the yield observed for C. tropicalis ATCC 20215 after growth for 120 h at 30 °C.     

Regeneration of plants from seed-derived callus of Hybanthus enneaspermus L. Muell., a rare ethnobotanical herb

A procedure was developed for plant regeneration of Hybanthus enneaspermus, a rare ethnobotanical herb from the Deccan peninsula in India, through seed-derived callus. Seeds demonstrated a high induction frequency (69.4±2.8%) and a high yield (364.4±2.5 mg) of light-yellow friable callus on Murashige and Skoog's (MS) medium containing 2.6 μm NAA and 2.2 μm BA within 4 weeks of incubation. After 1 year of subculture, yellow friable and light-green compact calli types were established from initial light-yellow friable callus. Shoot differentiation was achieved from light-green compact callus, but not from yellow friable callus. Shoot differentiation resulted when light-green compact callus was transferred to MS medium supplemented with 8.8 μm BA and 2.6 μm NAA; the highest percentage of calli forming shoots (66.6±4.8%) and the highest number of shoots (8.9±0.3) were achieved in this medium. Differentiated shoot buds elongated to 4–5 cm within 4 weeks. The addition of casein hydrolysate (500 mg/l) and more potassium phosphate (1.86 mm) to the culture medium enhanced shoot differentiation. Rooting was achieved on the shoots using half-strength MS medium containing 4.8 μm IBA. About 70% of the plants were established in pots containing pure garden soil after 2 weeks of hardening. The regenerated plants were morphologically uniform and exhibited normal seed set. Received: 23 July 1998 / Revision received: 18 November 1998 / Accepted: 26 November 1998     

Production of polyhydroxyalkanoates by <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> ATCC 17759 using a detoxified sugar maple hemicellulosic hydrolysate

Sugar maple hemicellulosic hydrolysate containing 71.9 g/l of xylose was used as an inexpensive feedstock to produce polyhydroxyalkanoates (PHAs) by Burkholderia cepacia ATCC 17759. Several inhibitory compounds present in wood hydrolysate were analyzed for effects on cell growth and PHA production with strong inhibition observed at concentrations of 1 g/l furfural, 2 g/l vanillin, 7 g/l levulinic acid, and 1 M acetic acid. Gradual catabolism of lower concentrations of these inhibitors was observed in this study. To increase the fermentability of wood hydrolysate, several detoxification methods were tested. Overliming combined with low-temperature sterilization resulted in the highest removal of total inhibitory phenolics (65%). A fed-batch fermentation exhibited maximum PHA production after 96 h (8.72 g PHA/L broth and 51.4% of dry cell weight). Compositional analysis by NMR and physical–chemical characterization showed that PHA produced from wood hydrolysate was composed of polyhydroxybutyrate (PHB) with a molecular mass (M _N) of 450.8 kDa, a melting temperature (T _m) of 174.4°C, a glass transition temperature (T _g) of 7.31°C, and a decomposition temperature (T _decomp) of 268.6°C.     

Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749

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

Production of extracellular water insoluble β-1,3-glucan (curdlan) fromBacillus sp. SNC07

β-1,3-Glucan (curdlan) is a water-insoluble polysaccharide composed exclusively of β-1,3 linked glucose residues. Extracellular curdlan was mostly synthesized byAgrobacterium species andAlcaligenes faecalis under nitrogen-limiting conditions. In this study, we screened the microorganisms capable of producing extracellular curdlan from soil samples. For the first time, we reported Gram-positive bacteriumBacillus sp. SNC 107 capable of producing extracellular curdlan in appreciable amounts. The effect of different carbon sources on curdlan production was studied and found that the yield of curdlan was more when glucose was used as carbon source. It was also found that maximum production was achieved when the initial concentration of ammonium and phosphate in the medium was 0.5 and 1.9 g/L respectively. In this study the curdlan production was increased from 3 to 7 g/L in shake flask cultures.     

An increase of curdlan productivity by integration of carbon/nitrogen sources control and sequencing dual fed-batch fermentors operation

Curdlan is produced by Agrobacterium sp. ATCC 31749 under nitrogen-limited conditions not associated with cell growth. A novel curdlan production process was developed based on the different nutrient requirements for microbial cell growth and its efficiency was increased by integrating carbon/nitrogen sources control and sequencing dual fed-batch fermentors operation. By feeding ammonium solution to supply abundant nitrogen source and controlling pH in Fermentor I, cell growth was accelerated. High cell density of 29 g/L was attained. The culture broth in Fermentor I was then inoculated into sequencing Fermentor II which alleviated the high requirement for dissolved oxygen and accumulation of inhibitory metabolic by-products during curdlan production. Fermentor I promoted cell growth. Curdlan production started instantaneously in Fermentor II. By feeding nutrient solution with high carbon/nitrogen ratio and NaOH solution for pH adjustment, a feasible and optimal curdlan production process was formulated. The productivity, conversion efficiency and curdlan yield were achieved of 0.98 g/(L h), 57% (w) and 67 g/L, respectively. Such novel process can be scaled up for significant cost reduction at the industrial level.     

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.