Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

Identification of Flavone Compounds in Eighteen Gramineae Species

Conditions for tryptophan synthesis from pyruvic acid, indole and NH₄Cl by Enterobacter aerogenes AHU 1540 having a high tryptophanase activity, were investigated using a reaction mixture containing 1.7% of pyruvic acid. Under optimum conditions, 16.4g/liter of tryptophan was accumulated after 24 hr of incubation.

Agaricus campestris AHU 9382 produced pyruvic acid in amounts of 22 ~ 26.5 g/liter from 5% of glucose after 3-days shaking culture. When E. aerogenes was added to this fermentation broth together with indole and NH₄Cl, pyruvic acid produced was rapidly converted to tryptophan and yields of tryptophan as high as 15 g/liter were obtained after 12 hr of incubation. Furthermore, pyruvic acid fermentation by Saccharomyces exiguus AHU 3110 or Corynebacterium sp. 37-3A could also be used as a pyruvic acid source for subsequent tryptophan production.     

Butyric acid production from brown algae using <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> ATCC 25755

Butyric acid fermentation by Clostridium tyrobutyricum ATCC 25755 using glucose or brown algae as a carbon source was carried out. Initially, different fermentation modes (batch, fed-batch, and semi-continuous) at pH 6 and 37°C were compared using a model medium containing glucose as a carbon source. By feeding the whole medium containing 40 ∼ 50 and 30 g/L of glucose into the fed-batch and semi-continuous fermentations, very similar butyrate yields (0.274 and 0.252 g butyrate/g glucose, respectively) and productivities (0.362 and 0.355 g/L/h, respectively) were achieved. The highest butyrate concentration was about 50 g/L, which was observed in the fed-batch fermentation with whole medium feeding. However, semi-continuous fermentation sustained a longer fermentation cycle than the fed-batch fermentation due to end-product and metabolic waste inhibition. The established conditions were then applied to the fermentation using brown algae, Laminaria japonica and Undaria pinnatifida, as substrates for butyric acid fermentation. To hydrolyze brown algae, 7.5 ∼ 10% (w/v) dried brown algae powder was suspended in 1% (w/v) NaOH or 0.5 ∼ 2.5% (w/v) H₂SO₄ and then autoclaved at 121°C for 30 ∼ 90 min. The resulting butyrate concentration was about 11 g/L, which was produced from 100 g/L of L. japonica autoclaved for 60 min in 1.5% H₂SO₄ acid solution.     

Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems

Rapid fermentation of bagasse hydrolysate to ethanol under anaerobic conditions by a strain of Saccharomyces cerevisiae has been studied in batch and continuous cultures at pH 4.0 and 30°C temperature with cell recycle. By using a 23.6 g/liter cell concentration, a concentation of 9.7% (w/v)ethanol was developed in a period of 6 hr. The rate of fermentation was found to increase with supplementation of yeast vitamins in the hydrolysate. In continuous culture employing cell recycle and a 0.127 v/v/m air flow rate, a cell mass concentration of 48.5 g/liter has been achieved. The maximum fermentor productivity of ethanol obtained under these conditions was 32.0 g/liter/hr, which is nearly 7.5 times higher than the normal continuous process without cell recycle and air sparging. The ethanol productivity was found to decrease linearly with ethanol concentration. Conversion of glucose in the hydrolysate to ethanol was achieved with a yield of 95 to 97% of theoretical.     

Heterotrophic Growth and Production of Xanthophylls by Chlorella pyrenoidosa

The growth and level of xanthophylls of several representative species of green algae were investigated as a possible source of pigmentation for the egg yolk and broiler markets. Chlorella pyrenoidosa 7-11-05 was selected for fermentation studies because of its high level of xanthophylls and wide temperature range for growth. The heterotrophic metabolism was preferred because of the ease of adaptability to present fermentation equipment. When used as the sole carbon source, glucose was the only sugar, among many tested, that gave appreciable growth in illuminated shaken flasks. A dry cell weight of 90 g per liter and total xanthophylls of 450 mg per liter were obtained from 190 g per liter of glucose monohydrate in 168-hr illuminated shaken flasks. Higher levels of glucose decreased yields. In combination with glucose, monosaccharides, such as fructose and galactose, were readily assimilated. The 7-11-05 strain was adapted to galactose as the sole carbon source after six vegetative passages. Light of the proper intensity and duration stimulated total xanthophylls approximately 35%. The effect on dry cell weight and total xanthophylls of seven antibiotics added at various levels in shaken flasks was studied. Erythromycin was essentially stable throughout the fermentation and nontoxic up to 25 μg/ml, with only slight toxicity at higher levels. Both erythromycin and ristocetin were effective in controlling a high incidence of bacterial contamination in 30-liter fermentors. With the higher agitation and aeration rates possible in 30-liter fermentors, dry cell weights in excess of 100 g per liter and total xanthophylls of 467 to 512 mg per liter were readily obtained from 230 to 260 g per liter of glucose in 162-hr illuminated batch-type fermentations. Continuous-feed runs yielded a dry cell weight of 302 g per liter and total xanthophylls of 650 mg per liter from 520 g per liter of glucose. The type of Chlorella cell produced was an important consideration with respect to the availability of the xanthophylls in pigmenting egg yolks and broilers.     

Simultaneous Production and Recovery of Fumaric Acid from Immobilized Rhizopus oryzae with a Rotary Biofilm Contactor and an Adsorption Column

An integrated system of simultaneous fermentation-adsorption for the production and recovery of fumaric acid from glucose by Rhizopus oryzae was investigated. The system was constructed such that growing Rhizopus mycelia were self-immobilized on the plastic discs of a rotary biofilm contactor during the nitrogen-rich growth phase. During the nongrowth, production phase, the biofilm was alternately exposed to liquid medium and air upon rotation of the discs in the horizontal fermentation vessel. The product of fermentation, fumaric acid, was removed simultaneously and continuously by a coupled adsorption column, thereby moderating inhibition, enhancing the fermentation rate, and sustaining cell viability. Another beneficial effect of the removal of fumaric acid is release of hydroxyl ions from a polyvinyl pyridine adsorbent into the circulating fermentation broth. This moderates the decrease in pH that would otherwise occur. Polyvinyl pyridine and IRA-900 gave the highest loading for this type of fermentation. This fermentation system is capable of producing fumaric acid with an average yield of 85 g/liter from 100 g of glucose per liter within 20 h under repetitive fed-batch cycles. On a weight yield basis, 91% of the theoretical maximum was obtained with a productivity of 4.25 g/liter/h. This is in contrast to stirred-tank fermentation supplemented with calcium carbonate, whose average weight yield was 65% after 72 h with a productivity of 0.9 g/liter/h. The immobilized reactor was operated repetitively for 2 weeks without loss of biological activity.     

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation

Propionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54?±?0.023?g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48?hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74?±?0.06?g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.     

Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

Enhanced Butanol Production by Clostridium beijerinckii BA101 Grown in Semidefined P2 Medium Containing 6 Percent Maltodextrin or Glucose

Dramatically elevated levels of butanol and acetone resulted in higher butanol and total solvent yields for hyperamylolytic Clostridium beijerinckii BA101 relative to the NCIMB 8052 parent strain grown in semidefined P2 medium containing either 6% glucose or STAR-DRI 5 maltodextrin. C. beijerinckii BA101 consistently produced on the order of 19 g of butanol per liter in 20-liter batch fermentations. This represents a greater than 100% increase in butanol concentration by the BA101 strain compared to the parent NCIMB 8052 strain. The kinetics of butanol production over time also indicate a more rapid rate of butanol production by BA101 in semidefined P2 medium containing glucose or maltodextrin. The lower levels of butyric and acetic acids produced over the course of the fermentation carried out by BA101 are consistent with an enhanced capacity for uptake and recycling of these acids. C. beijerinckii BA101 appears to more completely utilize carbohydrate compared to the 8052 strain. Carbon balance following fermentation by C. beijerinckii 8052 and BA101 indicates that sufficient carbon is available for the twofold increase in butanol concentration observed during BA101 fermentations. C. beijerinckii BA101 also has superior solvent production capacity during continuous culture fermentation in P2 medium containing 6% glucose. Volumetric solvent yields of 0.78 and 1.74 g/liter/h for BA101 and 0.34 and 1.17 g/liter/h for NCIMB 8052 were obtained at dilution rates of 0.05 and 0.20 h(sup-1), respectively. No drift towards acid synthesis (strain degeneration) was observed for up to 200 h (d = 0.05 h(sup-1)) and 100 h (d = 0.20 h(sup-1)).     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

The acid hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol sulfuric acid per liter pyrolysate using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol in 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol in 18 h. The results showed that the acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by S. cerevisiae (R) in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Sugar Substrates for l-Lysine Fermentation by Ustilago maydis

The extracellular production of l-lysine in media with cane sugar, blackstrap molasses, or clarified sugar-cane juice by a previously obtained mutant of Ustilago maydis was studied. Enzymatically inverted clarified juice (medium J-3) gave 2.9 g of lysine per liter under the following conditions: inoculum, 5%; pH 5.8; temperature, 30 C; K(La) in the fermentors, 0.41 mmoles of O(2) per liter per min; fermentation time, 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content.     

Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO₂-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.     

Acetone and Butanol Production by Clostridium acetobutylicum in a Synthetic Medium

The effect of the component concentrations of a synthetic medium on acetone and butanol fermentation by Clostridium acetobutylicum ATCC 824 was investigated. Cell growth was dependent on the presence of Mg, Fe, and K in the medium. Mg and Mn had deleterious effects when in excess. Ammonium acetate in excess caused acid fermentation. The metabolism was composed of two phases: an acid phase and a solvent one. Low concentrations of glucose allowed the first phase only. The theoretical ratio of the conversion of glucose to solvents, which was 28 to 33%, was obtained with the following medium: MgSO₄, 50 to 200 mg/liter; MnSO₄, 0 to 20 mg/liter; KCl, 0.015 to 8 g/liter (an equivalent concentration of K⁺ was supplied in the form of KH₂PO₄ and K₂HPO₄); FeSO₄, 1 to 50 mg/liter; ammonium acetate, 1.1 to 2.2 g/liter; para-aminobenzoic acid, 1 mg/liter; biotin, 0.01 mg/liter; glucose, 20 to 60 g/liter.     

Modelling and off line optimization of batch gluconic acid fermentation

Summary The role of mathematical modelling and off line optimization for a batch fermentation process is described. The fermentation of gluconic acid by Acetobacter suboxydans ATCC 621 was studied. The model is based on a series of batch experiments in which the temperature was the only variable. The differential equations of the models were derived from these experiments to give the kinetic parameters and the parametric models varying with the temperature. The fermentation was optimized using Pontryagin's maximum principle. This gave the temperature profile of fermentation.Abbreviations x, g, l, S, c, P The concentration of cell mass, glucose, lactone, gluconic acid, 5-ketogluconic acid and total acidic products respectively - r₁, r₂ E₁ or E₂ enzyme in complex/total E₁ or E₂ enzyme content - a, b E₁ and E₂ enzyme content of unit quantity of biomass - k_i and K_j Rate constants - _max Maximum specific growth rate - y_x z₁=r₁x z₂=r₂x Yield coefficient of biomass with respect to growth on glucose - z₁ r₁x - z₂ r₂x     

Production of microbial protein from tree bark by Phanerochaete chrysosporium

Phanerochaete chrysosporium was grown in fermentors on NaOH-extracted maple, pine, and cedar barks at the optimum substrate concentration of 1% (w/v). The yields (mg protein/liter) on maple, pine, and cedar were 1500, 1200, and 880, respectively, which are probably due to the different lignin contents of the barks. Lignin is not utilized. The productivities at 30°C obtained for pine (4.07 × 10^?2 g protein/liter hr) and cedar (2.63 × 10^?2 g protein/liter hr) barks were greater than for maple (2.63 × 10^?2 g protein/liter hr). The substrate (bark) was the limiting component of the fermentation. Over the 26–38°C temperature range protein productivity increased by a factor of three (1.55 × 10^?2 vs. 4.61 × 10^?2 g protein/liter hr) for maple bark. Low agitation rates resulted in an overproduction of cellulase and reduced levels of microbial protein.     

基于pH的反馈补料方法在谷氨酸发酵中的应用

为简化谷氨酸发酵补料工艺,提出了一种新型的基于pH的补料方式。考察谷氨酸发酵过程中氨消耗量 (x) 和糖消耗量 (y) 发现,两者之间存在较好的线性关系 (y=7.4744x,R2=0.9989),以此为pH反馈补料工艺中补料液中葡萄糖与氨的混合比例,能较好地将谷氨酸发酵过程中葡萄糖浓度稳定在12~21 g/L。比较恒定葡萄糖浓度补料工艺与pH反馈补料工艺发现,采用pH反馈补料工艺进行发酵,葡萄糖转化率、谷氨酸产酸速率分别提高了9.06％和17.5％左右,同时发酵周期缩短2 h以上。     

Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO2-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.     

Induction of Mutants from the Tartrate Producing Bacterium,Gluconobacter suboxydans and their Properties

The purpose of the present investigation is to obtain the superior mutants from the tartrate producing strain, Gluconobacter suboxydans 2026Y2 previously isolated from nature. Some mutant strains obtained by treatment with N-methyl-N′-nitro-N-nitrosoguanidine were found to accumulate L(+) tartaric acid in culture broth with much higher yield than in the case of the wild strain.

The high tartrate productivity of the mutants was followed by the low accumulation of 2-ketogluconic acid. The mutants having high assimilability of 5-ketogluconate showed high tartrate productivity.

The culture conditions for tartaric acid production by a mutant, Gl. suboxydans N-3874, were investigated. As a result, the amount of tartaric acid accumulated in culture broth reached to a level of 14.6g/liter in the medium containing 5% glucose and 0.3% corn steep liquor.     

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and its nutritive analysis

After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0°C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C_16:0 (19.0%), C_18:0 (46.3%), and C_18:1 (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production.     

Flocculant production from kerosene

Cultivation of Corynebacterium hydrocarboclastus, which is capable of synthesizing an extracellular polymer and utilized hydrocarbons, has been reported. Growth studies in shake flasks and fermenters were made to obtain maximum polymer production. Polymer formation was found to be growth associated. The highest level of polymer accumulation was attained after 50–60 hr cultivation in the fermenter and it amounted to approximately 5.5–6 g/liter of fermentation broth. The medium contained initially 2% (v/v) kerosene as a carbon source. The maximum yield obtained corresponds to 37–40% (w/w) of kerosene supplied. At the same time the cell concentration was 10–13 g/liter which represents the yield of 67–87% (w/w). The rate of polymer production in the exponential phase was 0.25 g/liter hr and cell production rate was 0.27 g/liter hr. Sodium nitrate, 0.5%, and yeast extract, 0.3%, (w/w) were the best nigrogen sources for polymer formation. The highest level of polymer produced in broth was 6 g/liter.