Optimal pH control of batch processes for production of curdlan by Agrobacterium species

We sought an optimal pH profile to maximize curdlan production in a batch fermentation of Agrobacterium species. The optimal pH profile was calculated using a gradient iteration algorithm based on the minimum principle of Pontryagin. The model equations describing cell growth and curdlan production were developed as functions of pH, sucrose concentration, and ammonium concentration, since the specific rates of cell growth and curdlan production were highly influenced by those parameters. The pH profile provided the strategy to shift the culture pH from the optimal growth condition (pH 7.0) to the optimal production one (pH 5.5) at the time of ammonium exhaustion. By applying the optimal pH profile in the batch process, we obtained significant improvement in curdlan production (64 g L⁻¹) compared to that of constant pH operation (36 g L⁻¹). Received 24 November 1998/ Accepted in revised form 17 June 1999     

Effective production of biologically active water-soluble β-1,3-glucan by a coupled system of Agrobacterium sp. and Trichoderma harzianum

Water-soluble β-1,3-glucan (w-glucan) prepared from curdlan is reported to possess various bioactive and medicinal properties. To develop an efficient and cost-effective microbial fermentation method for the direct production of w-glucan, a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT) was established. The effects of Tween-80, glucose flow rate, and the use of a dissolved oxygen (DO) control strategy on w-glucan production were assessed. The addition of 10?g?L^?1 Tween-80 to the CFS-AT enhanced w-glucan production, presumably by loosening the curdlan ultrastructure and increasing the efficiency of curdlan hydrolysis. A two-stage glucose and DO control strategy was optimal for w-glucan production. At the T. harzianum cell growth stage, the optimal glucose flow rate and agitation speed were 2.0?g?L^?1 hr^?1 and 600?rpm, respectively, and at the w-glucan production stage, they were 0.5?g?L^?1 hr^?1 and 400?rpm, respectively. W-glucan production reached 17.31?g?L^?1, with a degree of polymerization of 19–25. Furthermore, w-glucan at high concentrations exhibited anti-tumor activity against MCF-7, HepG2, and Hela cancer cells in vitro. This study provides a novel, cost-effective, eco-friendly, and efficient microbial fermentation method for the direct production of biologically active w-glucan.     

Residual phosphate concentration under nitrogen-limiting conditions regulates curdlan production in Agrobacterium species

We investigated the influence of inorganic phosphate concentration on the production of curdlan by Agrobacterium species. A two-step culture method was employed where cells were first cultured, followed by curdlan production under nitrogen-limiting conditions. In the curdlan production step, cells did not grow but metabolized sugar into curdlan. Shake-flask experiments showed that the optimal phosphate concentration for curdlan production was in the range of 0.1–0.3 g l⁻¹. As the cell concentration increased from 0.42 to 1.68 g l⁻¹ in shake-flask cultures, curdlan production increased from 0.44 to 2.80 g l⁻¹. However, the optimal phosphate concentration range was not dependent upon cell concentration. The specific production rate was about 70 mg curdlan g-cell⁻¹ h⁻¹ irrespective of cell concentration. When the phosphate concentration was maintained at 0.5 g l⁻¹ under nitrogen-limiting conditions, as high as 65 g l⁻¹ of curdlan was obtained in 120 h. Journal of Industrial Microbiology & Biotechnology (2000) 25, 180–183. Received 25 October 1999/ Accepted in revised form 21 July 2000     

Influence of pH on growth and bacteriocin production byLactococcus lactis subsp.lactis 14ONWC during batch fermentation

The influence of pH on growth, and lactic acid and bacteriocin production byLactococcus lactis subsp.lactis 140 NWC was studied during batch fermentation in a lactose-based complex medium. Growth and lactic acid production were modelled using a simple logistic equation while substrate consumption was found to be a function growth and lactic acid production rate. The optimal pH for growth and lactic acid production was between 6.0 and 6.5. Bacteriocin production showed primary metabolite kinetics. pH had a dramatic effect on the production of the bacteriocin, lactococcin 140. A maximum activity of 15.4 × 10⁶ AU (arbitrary units) 1^–1 was obtained after 7 h at pH 5.5. Maximum bacteriocin activity was achieved before the end of growth and was followed by a decrease in activity, which was due to adsorption to the cells of the producing organism, possibly followed by degradation by specific proteases. Both bacteriocin production and degradation rates were higher at pH 5.0 and 5.5, resulting in sharper activity peaks than at pH 6.0 or 6.5. On the basis of the experimental results a qualitative model for bacteriocin production is proposed.     

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.     

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).     

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.     

Production of curdlan using sucrose or sugar cane molasses by two-step fed-batch cultivation of Agrobacterium species

Maltose and sucrose were efficient carbon sources for the production of curdlan by a strain of Agrobacterium sp. A two-step, fed-batch operation was designed in which biomass was first produced, followed by curdlan production which was stimulated by nitrogen limitation. There exists an optimal timing for nitrogen limitation for curdlan production in the two-step, fed-batch operation. Maximum curdlan production (60 g L⁻¹) was obtained from sucrose with a productivity of 0.2 g L⁻¹ h⁻¹ when nitrogen was limited at a cell concentration of 16.0 g L⁻¹. It was also noted that the curdlan yield from sucrose was as high as 0.45 g curdlan g⁻¹ sucrose, and the highest specific production rate was 1.0 g curdlan g⁻¹ cells h⁻¹ right after nitrogen limitation. Of particular importance was the use of molasses as a cheap carbon source to produce curdlan in the two-step, fed-batch cultivation. As high as 42 g L⁻¹ of curdlan with a yield of 0.35 g curdlan g⁻¹ total sugar was obtained after 120 h of fed-batch cultivation. Received 20 August 1996/ Accepted in revised form 26 November 1996     

氮源NH4Cl浓度对粪产碱杆菌发酵生产热凝胶的影响

研究了利用粪产碱杆菌(Alcaligenes faecalis)发酵生产热凝胶的发酵条件,氮源是菌体生长的限制性底物,单纯地提高初始底物(氮源)浓度并不一定能促进细菌的生长和产物的合成.在分批发酵过程中,底物消耗导致培养环境pH的改变也是影响细菌进一步生长和产物合成的重要因素.通过增加培养基中初始氯化铵的浓度并同时控制发酵过程的pH条件,得到了较高的菌体浓度,热凝胶的合成水平也得到了显提高.当培养基中NH4Cl浓度提高到3.6g/L时,菌体浓度达到7.2g/L,热凝胶合成的产量可达30.5g/L,比原来NH4Cl浓度为1.1g/L时提高了51.7%.提高菌体浓度意味着需要提高溶氧水平来满足细菌的生长和代谢.初始氮源NH4Cl浓度的增加虽然能使菌体浓度得到提高,但发酵过程对溶氧的需求也相应增加,需要提高搅拌转速和通风以增加供氧水平.但高搅拌速率产生的高剪切力对热凝胶的凝胶性能将产生破坏作用,因此在发酵过程中需要综合考虑细菌培养密度对合成热凝胶产量和质量的影响.     

Effect of pH on growth and ethanol production byZymomonas

Summary In a mineral salts medium containing yeast extract, NH₄Cl and glucose (50g/L), the pH range producing the fastest growth ofZ. mobilis was 5.5–6.5 with an apparent optimum at 6.5. At constant growth rate of 0.15hr^–1, the specific rates of glucose utilization (q_s) and ethanol production (q_p) were relatively unaffected by pH over the range 7.0–5.5 but increased sharply as the pH was further decreased below 5.5 to 4.0. Under these conditions the ethanol yield was unaffected by pH over the range 4.0–6.5 but decreased markedly at pH of 7.     

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.     

pH控制对热凝胶发酵的影响

热凝胶 (Ｃｕｒｄｌａｎ)是一种直链结构的 β 1,3 葡聚糖 ,由Ａｌｃａｌｉｇｅｎｅｓｆａｅｃａｌｉｓｖａｒ.ｍｙｘｏｇｅｎｅｓ发酵生产而来 ,是一种新型的微生物胞外多糖[1 ] ,其分子量在 5 0万左右。热凝胶在中性条件下不溶于水 ,但能溶于碱溶液中。加热含有热凝胶的水浊液可形成两种类型的凝胶 ,一种是弹性较低的类似琼脂的可逆胶 ;另外一种是凝胶强度大、弹性好的热不可逆胶。由于热凝胶具有独特的热成胶性能 ,在食品工业 ,特别是高温制作的食品领域具有广阔的应用前景。热凝胶的胶体可以包容和控制药物的扩散 ,所以可以用来作为药物…     

枯草芽孢杆菌(Bacillus subtilis)发酵生产乙偶姻的pH调控策略

【目的】为了提高Bacillus subtilis CCTCC M 208157发酵生产乙偶姻的效率。【方法】在7 L发酵罐水平上考察不同pH条件对菌株生长及乙偶姻合成的影响。【结果】pH对菌株合成乙偶姻有显著影响,pH 4.5有利于细胞合成乙偶姻,但是延迟期较长;pH 5.5时菌株生长较快,但乙偶姻的产量偏低。因此提出了两阶段pH控制策略:发酵前期(0 16 h),控制pH 5.5;发酵中后期(16 72 h),控制pH 4.5。【结论】通过此策略,菌株合成乙偶姻的能力得到进一步提高,乙偶姻的产量、产率和生产强度分别为32.7 g/L、0.41 g/g和0.91 g/(L.h),分别比初始发酵条件下提高了41%、42%和69%。     

Production of tetramethylpyrazine by batch culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> with optimal pH control strategy

The effects of initial culture pH ranging from 5.0 to 7.5 on biomass content, precursor 3-hydroxy-2-butanone (HB) accumulation, and 2,3,5,6-tetramethylpyrazine (TTMP) formation by Bacillus subtilis CCTCC M 208157 were investigated in shake flask fermentation. Weak acidic conditions were found to favor cell growth and precursor HB accumulation, while TTMP could be synthesized more efficiently in conditions with initial pH towards neutrality. Batch bioprocess of TTMP fermentation by Bacillus subtilis CCTCC M 208157 at various controlled pH values ranging from 5.5 to 7.0 was then examined in 7.5-l fermentor. The results suggested that optimum pH for cell growth and precursor HB accumulation was 5.5 with maximum cell growth rate (Q _x) and precursor HB accumulation rate (Q _HB) of 0.833 g l⁻¹ h⁻¹ and 1.118 g l⁻¹ h⁻¹, respectively, while optimum pH for TTMP formation was 7.0 with maximum TTMP formation rate (Q _TTMP) of 0.095 g l⁻¹ h⁻¹. A pH-shifted strategy was accordingly developed to improve TTMP production in bioreactor fermentation by shifting the culture pH from 5.5 to 7.0 after 48 h of cultivation. By applying the strategy, final TTMP concentration of 7.43 g l⁻¹ was obtained, being 22.2% greater than that of constant-pH fermentation.     

Effect of environmental factors and precursors on oxalic acid production,mycelial biomass and virulence of a potential bioherbicide isolate of Sclerotium rolfsii SC64 produced in liquid culture

The fungus Sclerotium rolfsii is presently under development as a bioherbicide for broadleaf weed species using fungus-infested substrates as application material in this laboratory. The effect of environmental factors and three precursors (citric acid, ascorbic acid, and sodium succinate) on mycelial growth, oxalic acid production, and virulence by SC64 in liquid culture were investigated. The results showed that for mycelia growth the optimum liquid medium was Modified Richard's solution (MRS) among the five tested media, but potato dextrose broth (PDB) produced the maximum oxalic acid production and virulence on detached Solidago canadensis leaves. When PDB was used as the basic medium, the oxalic acid/mycelial dry weight (mg g^–1) ratio reached the peak 4 days after inoculation. The optimum temperature for oxalic acid production was at 27°C, but increased mycelial dry weight and virulence were observed at 30°C. The optimum range of initial pH value for oxalic acid accumulation was 4.0–6.0, with the optimal pH 5.0; highest mycelial growth was with an initial pH 3.5–6.0 (optimum pH 5.0) and subsequently pH 3.5–5.5 (maximum at pH 3.5). Both mycelial dry weight and oxalic acid production showed a decreasing trend as a result of the precursor of oxalic acid being added to PDB. Among the three precursors, the greatest decrease in mycelial dry weight, and oxalic acid production was caused by sodium succinate. This clarification of optimal conditions for production of mycelial biomass while insuring high concentrations of oxalic acid and high virulence should be useful for further development of this fungus as biocontrol agent.     

Improved production of polygalacturonate lyase by combining a pH and online methanol control strategy in a two-stage induction phase with a shift in the transition phase

Polygalacturonate lyase is a kind of enzyme that is abundantly used in the textile industry for cotton scouring. Previously, we reconstructed the polygalacturonate lyase gene in Pichia pastoris for the expression of this enzyme. To enhance the production of polygalacturonate lyase (PGL), a combined strategy was formulated by combining online methanol control and two-stage pH control strategies. For the two-stage pH control strategy during the growth phase, the pH was controlled at 5.5, and in the induction phase different pH levels were investigated for the optimum enzyme production. During the online methanol control strategy, the different levels of methanol (v/v) were investigated for the best enzyme production at pH 5.5. These two strategies were combined together for enhanced PGL productivity, and the induction phase was divided into two stages in which methanol concentrations were maintained at different levels online. The transition phase was introduced during the induction phase instead of introducing it after the growth phase. The two-stage combination strategy was formulated on the bases of methanol consumption of cells, optimal pH, cell viability and the production of polygalacturonate lyase by P. pastoris. By using this strategy, the production was doubled compared with common conditions, and the highest polygalacturonate lyase activity reached 1,631 U/ml. This strategy proved to be very useful for the enhancement of polygalacturonate lyase production by achieving higher cell viability, alcohol oxidase activity and phosphate-related compounds of the cells during the induction phase.     

Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: relationship between production of the lantibiotic and lactate and cell growth

 The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied. Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail. Production of nisin Z was optimal at 30°C and in the pH range 5.0–5.5. The addition of Ca²⁺ to the medium showed a stimulating effect on the production of nisin Z. A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl₂. It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production. Received: 12 July 1995/Received revision: 11 September 1995/Accepted: 4 October 1995     

氨水流加用于粪产碱杆菌热凝胶发酵

热凝胶是粪产碱杆菌(Alcaligenes faecalis)在氮源限制条件下生成的水不溶性胞外多糖,分泌到胞外后就附着在菌体外壁,因此在细胞生长期提高生物量对促进热凝胶合成有重要意义。热凝胶分批发酵时, 起始NH4Cl浓度提高到3.6 g/L时能促进菌体生长和热凝胶合成,但是过量NH4Cl会抑制热凝胶合成,且生物量提高不是很明显。为了进一步提高菌体浓度, 在菌体生长期, 氨水取代NaOH溶液进行流加控制pH为7.0, 随后又用2 mol/L NaOH控制pH 5.6。实验表明, 氨水流加使菌体浓度大大提高,流加24 h使菌体浓度达到18.8 g/L。但是菌体浓度过高也会抑制热凝胶的合成,在氨水流加14 h时,菌体浓度在11.9 g/L左右, 热凝胶产量最高（72 g/L）。     

Optimization of culture conditions for the production of an extracellular ribonuclease by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a benchtop bioreactor

SummarySelf-directing optimization was successfully employed to determine the optimal combination of engineering parameters, viz., pH, aeration rate and agitation rate, for extracellular ribonuclease production by Aspergillus niger SA-13-20 in a batch bioreactor. Maximal RNase production of 5.38 IU ml^–1 was obtained at controlled pH of 2.33, aeration rate of 1.67 v/v/m and agitation rate of 850 rev/min. The effect of oxygen on the fermentation was also investigated. With increase in volumetric oxygen transfer coefficients (K_La), cell growth and RNase production first increased and then decreased. RNase production was further increased to 7.10 IU ml^–1 and the fermentation time was shortened from 96 to 72 h by controlling dissolved oxygen concentration at 10% saturation by aerating oxygen after about 28 h of fermentation under the above optimal condition. The kinetic model showed that RNase production by A. niger SA-13-20 was growth-associated.     

氨水流加用于粪产碱杆菌热凝胶发酵

热凝胶是粪产碱杆菌(Alcaligenes faecalis)在氮源限制条件下生成的水不溶性胞外多糖,分泌到胞外后就附着在菌体外壁,因此在细胞生长期提高生物量对促进热凝胶合成有重要意义。热凝胶分批发酵时, 起始NH4Cl浓度提高到3.6 g/L时能促进菌体生长和热凝胶合成,但是过量NH4Cl会抑制热凝胶合成,且生物量提高不是很明显。为了进一步提高菌体浓度, 在菌体生长期, 氨水取代NaOH溶液进行流加控制pH为7.0, 随后又用2 mol/L NaOH控制pH 5.6。实验表明, 氨水流加使菌体浓度大大提高,流加24 h使菌体浓度达到18.8 g/L。但是菌体浓度过高也会抑制热凝胶的合成,在氨水流加14 h时,菌体浓度在11.9 g/L左右, 热凝胶产量最高（72 g/L）。