Citric acid production patent review.

Current Review article summarizes the developments in citric acid production technologies in East and West last 100 years. Citric acid is commercially produced by large scale fermentation mostly using selected fungal or yeast strains in aerobe bioreactors and still remains one of the runners in industrial production of biotechnological bulk metabolites obtained by microbial fermentation since about 100 years, reflecting the historical development of modern biotechnology and fermentation process technology in East and West. Citric acid fermentation was first found as a fungal product in cultures of Penicillium glaucum on sugar medium by Wehmer in 1893. Citric acid is an important multifunctional organic acid with a broad range of versatile uses in household and industrial applications that has been produced industrially since the beginning of 20(th) century. There is a great worldwide demand for citric acid consumption due to its low toxicity, mainly being used as acidulant in pharmaceutical and food industries. Global citric acid production has reached 1.4 million tones, increasing annually at 3.5-4.0% in demand and consumption. Citric acid production by fungal submerged fermentation is still dominating, however new perspectives like solid-state processes or continuous yeast processes can be attractive for producers to stand in today's strong competition in industry. Further perspectives aiming in the improvement of citric acid production are the improvement of citric acid producing strains by classical and modern mutagenesis and selection as well as downstream processes. Many inexpensive by-products and residues of the agro-industry (e.g. molasses, glycerin etc.) can be economically utilized as substrates in the production of citric acid, especially in solid-state fermentation, enormously reducing production costs and minimizing environmental problems. Alternatively, continuous processes utilizing yeasts which reach 200-250 g/l citric acid can stand in today's strong competition in citric acid industry and replace the traditional discontinuous fungi processes.

L-lysine fermentation.

Amino acids are the basic bioelements of proteins, which are the most important macromolecules for the functions of humans and animals. Out of the 20 L-amino acids, ecumenically found in most of living organisms, L-lysine is one of the 9 amino acids which are essential for human and animal nutrition. L-lysine is useful as medicament, chemical agent, food material (food industry) and feed additive (animal food). Its demand has been steadily increasing in recent years and several hundred thousands tones of L-lysine (about 800,000 tones/year) are annually produced worldwide almost by microbial fermentation. The stereospecificity of amino acids (the L isomer) makes the fermentation advantageous compared with synthetic processes. Mutant auxotrophic or resistant to certain chemicals strains of so-called gram positive coryneform bacteria are generally used, including the genera Brevibacterium and Corynebacterium, united to the genus. The significance of Research and Development increased rapidly since the discovery of fermentative amino acid production in the fifties (S. Kinoshita et al., Proceedings of the International Symposium on Enzyme Chemistry 2:464-468 (1957)), leading to innovative fermentation processes which replaced the classical manufacturing methods of L-lysine like acid hydrolysis. L-Lysine is separated and purified by suitable downstream processes involving classical separation or extraction methods (ultrafiltration or centrifugation, separation or ion exchange extraction, crystallization, drying) and is sold as a powder. Alternatively, spray dried pellets or liquid fermentation broth can be used as animal feed supplement. On behalf of today's strong competition in amino acid industry, Biotechnology companies are continuously aiming in innovative research developments and use complex management concepts and business strategies, towards gaining market leadership in the field of amino acid production.

Gluconic acid production.

Gluconic acid, the oxidation product of glucose, is a mild neither caustic nor corrosive, non toxic and readily biodegradable organic acid of great interest for many applications. As a multifunctional carbonic acid belonging to the bulk chemicals and due to its physiological and chemical characteristics, gluconic acid itself, its salts (e.g. alkali metal salts, in especially sodium gluconate) and the gluconolactone form have found extensively versatile uses in the chemical, pharmaceutical, food, construction and other industries. Present review article presents the comprehensive information of patent bibliography for the production of gluconic acid and compares the advantages and disadvantages of known processes. Numerous manufacturing processes are described in the international bibliography and patent literature of the last 100 years for the production of gluconic acid from glucose, including chemical and electrochemical catalysis, enzymatic biocatalysis by free or immobilized enzymes in specialized enzyme bioreactors as well as discontinuous and continuous fermentation processes using free growing or immobilized cells of various microorganisms, including bacteria, yeast-like fungi and fungi. Alternatively, new superior fermentation processes have been developed and extensively described for the continuous and discontinuous production of gluconic acid by isolated strains of yeast-like mold Aureobasidium pullulans, offering numerous advantages over the traditional discontinuous fungi processes.

Continuous gluconic acid production by Aureobasidium pullulans with and without biomass retention

New alternative processes for the continuous production of gluconic acid by Aureobasidium pullulans, using biomass retention by cell immobilization or cross over filtration, are described in the present work. 315 g/l gluconic acid was continuously produced in chemostat cultures at 21 hrs residence time without any biomass retention. 260 g/l gluconic acid was produced in fluidized bed reactor at 21 hrs residence time. The support carrier was overgrown resulting in limitations of oxygen transfer towards the inner layers of immobilized biomass. 375 g/l gluconic acid was produced under continuous cultivation at 22 hrs of residence time with a formation rate for the generic product of 17 g/(l x h) and a specific gluconic acid productivity of only 0.74 g/(g x h), using biomass retention by cross over filtration. 370 g/l were obtained at 19 hrs RT and 100 percent conversion with 25 g/l biomass and a formation rate of 19 g/(l x h). At 100 percent conversion, a selectivity of only 78 percent was determined at 22 hrs and of 77 percent at 19 hrs RT, because of the very high biomass concentration. Biomass retention makes it possible to break the existing link between growth and residence time.

Continuous gluconic acid production by the yeast-like Aureobasidium pullulans in a cascading operation of two bioreactors.

The application of a new developed process for the continuous production of gluconic acid using a cascade of two bioreactors in a continuous process is shown reaching the highest concentration of gluconic acid described in the literature for continuous culture fermentation. Very high gluconic acid concentrations of 272-308 g/l have been achieved under continuous cultivation of free-growing cells of Aureobasidium pullulans in the first bioreactor at residence times (RT) between 19.5 and 24 h with formation rates for the generic product between 12.7 and 13.9 g/(l h). Gluconic acid, 350-370 g/l, was continuously reached in the second bioreactor at a total RT of 30.8-37 h with R (j) of 9.2-12 g/(l h). The highest specific gluconic acid production (m (p)) of 3.6 g/(g h) was found in the first bioreactor at the lowest RT of 19.5 h. The highest selectivity of 93.6% was determined in the first bioreactor as well. Complete glucose consumption was obtained at 37 h total residence time in the second bioreactor. Gluconic acid, 433 g/l, was continuously produced in the second bioreactor at a total RT of 37 h.

Oxygen and temperature effect on continuous citric acid secretion in Candida oleophila

The influence of air saturation and temperature on continuous citric acid secretion was studied in chemostat cultures of Candida oleophila ATCC 20177 (var.). Simultaneous measurements of intra- and extracellular concentration of glucose, citric and isocitric acid confirmed the involvement of a specific active transport system in citrate secretion, favouring citric acid over isocitrate. An optimum air oxygen saturation of 20 percent and temperature of 30-31ºC were determined for the continuous citric acid secretion. The highest values of citric acid concentration (98 g/L), citrate to isocitrate ratio (33.3:1), volumetric citric productivity (1.8 g/(L x h)), and specific citric acid productivity (0.1 g/(g x h)), were reached at 20 percent air saturation at a residence time of 54 hrs by the experiment's lowest biomass of 18 g/L. The highest isocitic acid volumetric productivity (55.6 mg/(L x h)) and specific productivity (0.99 mg/(g x h)) were identified at 50 percent, instead. The fastest citrate excretion rate of the generic product of 0.046 g/(g*h) was found at 30-31ºC. A concentration ratio between extra- and intracellular concentration of citrate of up to 9 was identified. The highest extra-/intracellular ratio of citrate and lowest intracellular concentrations of glucose, citric and isocitric acid were determined at optimum air saturation as a consequence of active citrate export.

Citric acid production from glucose by yeast Candida oleophila ATCC 20177 under batch, continuous and repeated batch cultivation

The effect of air saturation on citric acid production in batch, repeated batch and chemostat cultures has been studied. It was shown that, under continuous fermentation (chemostat mode), the highest concentration of citric acid equal of 98 g/l was produced at 20% of air saturation. In contrary to continuous fermentation, displaying an optimum at 20%, 80% air saturation yielded higher values in repeated batch fermentation process. 167 g/l citric acid were produced continuously with the fill and drain technique at 4.85 days, at 80% air saturation, compared with 157.6 g/l achieved within 5.4 days at 20%. Under repeated batch fermentation, the formation rate of the generic product (Rj) as well as the specific citric acid productivity (mp) reached a maximum of 1.283 g/(l x hr) at 4.01 days and of 0.0375 g/(g x hr) at 4.58 days, respectively. The glucose consumption rate (Rx) reached a maximum value of 3.33 g/(l x hr) entering stationary phase after 2.56 days at a glucose concentration of 131.2 g/l.

Continuous citric acid secretion by a high specific pH dependent active transport system in yeast Candida oleophila ATCC 20177

The pH influence on continuous citric acid secretion was investigated in Candida oleophila ATCC 20177 (var.) under NH4+ limiting state steady conditions, using glucose. Highest citric acid concentration of 57.8 g/l, citrate/isocitrate ratio of 15.6, space-time yield of 0.96 g/(l x hr) and biomass specific productivity of 0.041 g/(g x hr) were obtained at pH 5 and 60 hrs residence time. Only 22.8 g/l (39.4%) and a ratio of 9.9 were achieved at pH 6 pH and 12.4 g/l (21.5%) and a ratio of 3.7 at pH 3. Under non producing conditions, in excess of nitrogen, biomass concentration increased at raising pH. An iron concentration of 200 ppm was determined in biomass of C. oleophila at pH 5, compared with only 26 ppm found at pH 3 (factor 7.7). Intra- and extracellular concentrations of citrates and glucose confirmed the existence of a high specific, pH dependent active transport system for citrate secretion, while isocitrate isn’t a high-affine substrate, displaying a strong correlation with ATP/ADP ratio. Differences between extra- and intracellular concentration of citrate higher than 1 and up to about 60 were determined. The active transport systemfor citrate excretion appears to be the main speed-determining factor in citrate overproduction by yeasts.

Process optimization of continuous gluconic acid fermentation by isolated yeast-like strains of Aureobasidium pullulans.

This study was focused on the optimization of a new fermentation process for continuous gluconic acid production by the isolated yeast-like strain Aureobasidium pullulans DSM 7085 (isolate 70). Operational fermentation parameters were optimized in chemostat cultures, using a defined glucose medium. Different optima were found for growth and gluconic acid production for each set of operation parameters. Highest productivity was recorded at pH values between 6.5 and 7.0 and temperatures between 29 and 31 degrees C. A gluconic acid concentration higher than 230 g/L was continuously produced at residence times of 12 h. A steady state extracellular gluconic acid concentration of 234 g/L was measured at pH 6.5. 122% air saturation yielded the highest volumetric productivity and product concentration. The biomass-specific productivity increased steadily upon raising air saturation. An intracellular gluconic acid concentration of about 159 g/L (0.83 mol) was determined at 31 degrees C. This is to be compared with an extracellular concentration of 223 g/L (1.16 mol), which indicates the possible existence of an active transport system for gluconic acid secretion, or the presence of extracellular glucose oxidizing enzymes. The new process provides significant advantages over the traditional discontinuous fungi operations. The process control becomes easier, thus offering stable product quality and quantity.