Appl Microbiol Biotechnol (2007) 75:713722 DOI 10.

1007/s00253-007-0851-x

MINI-REVIEW

Biotechnological production of gluconic acid: future implications

Om V. Singh & Raj Kumar

Received: 26 October 2006 / Revised: 16 January 2007 / Accepted: 21 January 2007 / Published online: 14 February 2007 # Springer-Verlag 2007

Abstract Gluconic acid (GA) is a multifunctional carbonic acid regarded as a bulk chemical in the food, feed, beverage, textile, pharmaceutical, and construction industries. The favored production process is submerged fermentation by Aspergillus niger utilizing glucose as a major carbohydrate source, which accompanied product yield of 98%. However, use of GA and its derivatives is currently restricted because of high prices: about US$ 1.208.50/kg. Advancements in biotechnology such as screening of microorganisms, immobilization techniques, and modifications in fermentation process for continuous fermentation, including genetic engineering programmes, could lead to costeffective production of GA. Among alternative carbohydrate sources, sugarcane molasses, grape must show highest GA yield of 95.8%, and banana must may assist reducing the overall cost of GA production. These methodologies would open new markets and increase applications of GA. Keywords Gluconic acid . Microbial fermentation . Glucose oxidase . Alternative carbohydrate sources . Biotechnology . System biology
Authors contributions OVS and RK are the sole contributors of this original review article. This review is based upon the published research in the area of gluconic acid fermentation. O. V. Singh (*) Department of Pediatrics, The Johns Hopkins School of Medicine, 600 N. Wolfe St., CMSC, 3-106, Baltimore MD 21287, USA e-mail: osingh1@jhmi.edu e-mail: ovs11@yahoo.com R. Kumar Division of Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, New Delhi 110 054, India

Introduction Because of rising interest in sustainable development, there is extensive demand in the construction, chemical, and pharmaceutical industries for an absolute polyhydroxycarboxylic acid that has both reactive hydroxyl and carboxyl groups. Gluconic acid (GA, penta-hydroxycaproic acid; Fig. 1) is a naturally occurring polyhydroxycarboxylic acid commonly found in humans and other organisms. GA has been manufactured so far by discontinuous, intermittent batch and fed batch processes. The low selectivity in chemical synthesis of GA is uneconomical for industrial purposes (Hustede et al. 1989). Hence, the microbial conversion of glucose into GA in submerged fermentation process employing fungal species like A. niger and Penicillium and bacterial species such as Pseudomonas, Acetobacter, and Gluconobacter, etc. has been reviewed in the past (Milson and Meers 1985, Ramachandran et al. 2006). The yeast-like strain Aureobasidium pullulans has been evaluated for GA production with success (Anastassiadis et al. 2003, 2005; Anastassiadis and Rehm 2006a,b). The uneconomical carbohydrate substrate glucose and the system-specific requirements in fermentation contribute to the high price of GA and its derivatives (Table 1). However, its huge market consumption (Table 2) has spurred interest in the development of an effective and economical system for GA production. Among its other outstanding properties, GA has an excellent chelating capacity in alkaline solutions. GA has been extensively used in the cleaning and construction industries as an additive to increase cement resistance and stability under extreme climatic conditions (Milson and Meers 1985; Roehr et al. 1996; Ramachandran et al. 2006). Thus, the enormous application-dependent growth of GA enhanced the total market value: about US$333 million (Table 3; Business Communication Co. 2004).

714 Fig. 1 Gluconic acid formula and some physical properties

Appl Microbiol Biotechnol (2007) 75:713722

The surplus demand is about 87,000 tonnes (Table 2; Business Communication Co. 2004), and an estimated higher cost of US$ 1.208.50/kg of GA and its derivatives (Table 1) can be conquered by exploring unconventional carbohydrate sources such as agro-food by-products and modifications to the fermentation process. A wide variety of cheaper carbohydrate sources including sugarcane molasses, beet molasses, grape must, banana must, and paper waste have been proposed as substrates for GA production with 8595% yield (Kundu and Das 1984; Roukas and Harvey 1988; Buzzini et al. 1993; Rao et al. 1994; Rao and Panda 1994; Singh et al. 2003, 2005; Ikeda et al. 2006; Singh and Singh 2006). The key question is whether it is possible to exploit the availability of alternative carbohydrate sources to develop an industrially feasible GA-production technology, despite the fact that the indigenous demand for GA is met using pure glucose substrate. Rather than summarize all of the existing literature on GA production using glucose, the present article deals with the crucial parameters for developing an indigenous technology for GA fermentation by exploring a variety of cheaper alternative carbohydrate sources. A critical set of fermentation processes suitable for continuous GA fermentation is also discussed.

Microorganisms: asset in GA fermentation! Microorganisms have excelled at producing primary and secondary metabolites from a variety of raw carbohydrates since billions of years, and one of them was alcoholic fermentation. In current vogue, the results of studying giant microbial libraries for microbial conversion of cheaper carbohydrates into value-added products can serve as a raw material for GA fermentation. In fungi such as A. niger, GA is a product of simple dehydrogenation catalyzed by glucose oxidase (GOX) from D-glucose (Fig. 2). This oxidation reaction of an aldehyde group forms a carboxylic group resulting in glucono-lactone and H2O2, which further hydrolyzes into GA

spontaneously or via a lactone-hydrolyzing enzyme decomposing H2O2 into water and oxygen (Fig. 2). Besides A. niger, other species of the genera such as Penicillium, Gliocadium, Scopulariopsis, and Gonatobotrys have been tested for GA production and reviewed by Milson and Meers (1985) and Ramachandran et al. (2006). Several bacterial species, including G. oxydans, Z. mobilis, A. methanolicus, P. fluorescens, and the species of Morexella, Tetracoccus, Pullularia, Micrococcus, Enterobacter, and Scopulariopsis participate in GA production with a specific pathway by oxidizing glucose into GA with glucose dehydrogenase (Fig. 2); this process has been reviewed by Milson and Meers (1985) and Ramachandran et al. (2006). In the past, a variety of microorganisms have been explored that can utilize the alternative carbohydrate sources (such as hydrol, corn starch, grape must, banana must, fig, cheese whey, food processing residues, and saccharified solution of waste paper) with an 8095% yield of GA in submerged and solid-state surface fermentation (Roukas and Harvey 1988; Buzzini et al. 1993; Roukas 2000; Mukhopadhyay et al. 2005; Singh et al. 2005; Ikeda et al. 2006; Singh and Singh 2006). A. niger was grown in concentrated, rectified grape must with 67.43 g/l GA and a yield of 96% (converted glucose; Buzzini et al. 1993), and 96.52 g/l with a 95% yield of GA (Singh and Singh 2006). A. niger ATCC 10577 was employed on fig fruit for GA and citric acid production with 490 g GA/kg dry fig
Table 1 Market value of gluconic acid and derivatives based upon major industrial applicability (Source: Business Communication Co., Inc. 2004) Major product and salts Gluconic acid Sodium gluconate Pure sodium gluconate Calcium gluconate and -lactone Applicability Average cost ($/kg) 1.20 2.00 3.00 8.50

Cement industries Cement industries Food industry Food and pharamaceutical industries

Appl Microbiol Biotechnol (2007) 75:713722 Table 2 World wide consumption of gluconic acid based on major industrial applications (Source: Business Communication Co., Inc. 2004) Usage Quantity (in tonnes) 8,000 30,000 40,000 9,000 87,000 Total consumption (%) 9.2 34.5 46.0 10.3 100.0

715

Pharmaceutical industries Food industries Construction industries Other usage Total

(Roukas 2000). Another strain of A. niger NCIM 548 was grown on deproteinised whey as a nutritive medium producing 92 g GA/l whey (Mukhopadhyay et al. 2005). The microorganism A. niger IAM 2094, which consumes glucose as a carbon source and produce GA (Sakurai et al. 1989; Sankpal et al. 1999; Sankpal and Kulkarni 2002), was employed in saccharified solution of waste papers and provided 92% yield of GA (Ikeda et al. 2006). A continuous and discontinuous fermentation process using A. pullulans offers a new opportunity for commercial GA production, with 504 g/l GA in fed batch and 400 g/l in chemostate operations produced by free-growing cells with a maximum of 96.7% yield (Anastassiadis et al. 2003). In support of this study, it is known that A. niger is difficult to handle because of its clogging tendency; no toxicity has been reported so far of formed GA in fermentation medium, whereas bacterial systems are more sensitive at high glucose concentrations. A. pullulans integrates the advantages of both fungal and bacterial systems, utilizing a higher glucose concentration in continuous operation to produce 350433 g/l GA (Anastassiadis and Rehm 2006b). However, the growth and GA-production efficiency of this microorganism over a wide range of complex carbohydrates is still unclear.

Evaluation of GA fermentation process (1) Fermentation mediumEach microorganism has its own adaptability on a variety of fermentation media. However, several essential nutrients are required for any fermentation reaction. A variety of constituents have been defined in the past for GA fermentation media with different microorganisms (Nakamatsu et al. 1975; Mahmoud et al. 1976; Singh et al. 2001b, Anastassiadis et al. 2005; Singh and Singh 2006). Herrick and May (1928) presented the first systematic study of the influence of medium composition on GA production by P. luteum purpunogenum group organisms. A critical analysis of salt concentration can

maximize GA production and cut down on contaminating side products generated from A. niger during the transformation process (Moksia et al. 1996). In addition to minor elements, media containing traces of magnesium, potassium, and phosphate salts are most suitable for A. niger with 8085 g/l GA production while exploring grape must as a carbohydrate source (Singh and Singh 2006). (2) Carbon sourcesThe smooth operation of any fermentation industry largely depends on the cost of manufacture, and this in turn depends on the selection of raw materials. Glucose at a concentration of 10 15%, either in the form of glucose monohydrate crystals or fructose/dextrose syrup, has long been proposed as the main carbon source for GA production, with a 9095% yield (Silveira et al. 1999; Singh et al. 2001a,b; Klein et al. 2002; Znad et al. 2004). However, because microorganisms can be grown on a wide variety of carbohydrate sources, the agro-food by-products can be considered as an economical source of carbohydrates for GA fermentation. Among the major agro-food by-products, sugarcane molasses and grape must have high sugar content and can be used for the production of alcohol, which makes them suitable candidates for the GA fermentation process. The alternative carbohydrate sources with the potential to make the GA fermentation process more economical are hydrol, corn starch, can molasses, grape must, banana must, food processing residues, figs, cheese whey, beet molasses, and saccharified solution of waste paper (Kundu and Das 1984; Roukas and Harvey 1988; Buzzini et al. 1993; Rao and Panda 1994; Roukas 2000; Fischer and Bipp 2005; Singh et al. 2005; Singh and Singh 2006; Ikeda et al. 2006). Grape must and concentrated, rectified grape must have been used for GA synthesis in batch cultures with a production of 67.43 g/l in 72 h (Buzzini et al. 1993). In another attempt using fig fruit, Roukas (2000) obtained 490 g GA/kg dry figa 63% yieldand found that adding modulators such as 6% methanol into substrate enhances the levels of GA (685 g GA/kg dry fig). A by-product of dairy industry, deproteinised whey containing 9.5% lactose
Table 3 Major application-based global market expenditure in past and future assumption of gluconic acid (Source: Business Communication Co., Inc. 2004) Application 2004 (US$ million) 60 150 63 55 333 2009 (US$ millions) 76 153 51 31 311 AAGR% 20042009 4.8 0.4 5.6 10.8 1.4

Construction Food Pharmaceuticals Others Total

716 Fig. 2 Enzymatic mechanism of microbial conversion of Dglucose in to gluconic acid and downstream products (FAD, Flavin adenine dinucleotide; TCA, Tri-carboxylic acid cycle)

Appl Microbiol Biotechnol (2007) 75:713722

and 0.5% glucose was used as a nutritive medium and resulted into 92 g/l GA production (Mukhopadhyay et al. 2005). About 92% GA yield was observed utilizing saccharified solution of waste papers (Ikeda et al. 2006). On another occasion, 95% yield was recorded in a semicontinuous production of GA under pseudoimmobilization of A. niger on HCl-treated bagasse fibers utilizing rectified grape must as the substrate (Singh and Singh 2006). However, before these substrates can be used economically, one limitation is that the crude form of sugarcane molasses and grape must contain varying content of heavy metal ions and needs to be eliminated before GA fermentation. Therefore, the clarification of sugars is an absolute requirement and one of the most challenging tasks. (3) Substrate clarificationEconomical rectification of carbohydrate sources while maintaining their nutrient composition is a major dilemma in the fermentation industries. Hexacyanoferrate (HCF) was first used in the Belgian factory of Kaznezow in 1919 to precipitate interfering substances in molasses during citric acid production (Rohr et al. 1983). A complete study on the effect of HCF on molasses medium was performed (Clark et al. 1966; Kundu et al. 1984; Rao et al. 1994; Singh 2000), showing that the HCF treatment effectively precipitates most of the heavy metals (>95%) without affecting the mediums nitrogen, carbon, and phosphorus content. More than 85% of metallic ions were removed using sephadex fractionation of Indian cane molasses treated with tricalcium phosphate with hydrochloric acid for citric acid production (Kundu et al. 1984). A significant increase in GA production from 12 to 60 g/l with 61% yield was achieved using HCF-clarified cane molasses (Singh et al. 2005). A mutant of A. niger showed a 3.43-fold increase in GOX levels (2.62 U/ ml) when HCF-treated cane molasses was used in the fermentation medium instead of the crude form of

cane molasses (0.762 U/ml; Singh 2006). Hence, the efficient clarification of raw sugars in the fermentation medium is prerequisite for GA fermentation. Horticultural industry by-products such as grape must and banana must also contain trace amounts of metallic ions (Singh 2000), with varying amounts of vitamins such as carotene, thiamine, riboflavin, and pentothenate. The comprehensive composition of crude grape must determined by Atomic Absorption Spectroscopy (AAS), appears in Table 4. In a study utilizing grape and banana must for GA production, an enzymatic clarification was performed using cytolase, klerzyme, and rapidase for grape and banana musts (Singh et al. 2005). The clarified grape juice was referred to as rectified grape must and diluted equivalent to 120 g/l glucose before being preceded for fermentation (Singh et al. 2005; Singh and Singh 2006). A 2025% increase in GA production was observed in submerged fermentation of A. niger when rectified grape must and banana must equivalent to 12% of glucose were
Table 4 Major composition of grape must (adopted from Singh 2000) Components Total carbohydrate Sodium Potassium Calcium Magnesium Phosphorus Iron Copper Zinc Manganese Waterc
a

In percenta 503.8; 954.8b 0.0100.0015 0.0720.03 0.0220.018 0.0080.002 0.0160.009 0.00030.0001 0.000010.000005 None None 81.55.3

Values are average of at least triplicate samples (SD) from at least 34 batches of grape extracts were analyzed using AAS. b Values calculated after rectification of substrate. c Water contents were by difference.

Appl Microbiol Biotechnol (2007) 75:713722 Table 5 Major US patents in last 10 years for gluconic acid/gluconate production processesa Patent number EP649899 BR9403981 WO9635800 WO9724454 5962286 6828130 6942997
a

717

Patent year 1995 1996 1996 1997 1999 2004 2005

Author

Patent process

Kiyoshi, A and Yosuo , M Jonas, R.H.H.H.; Moura-de-Silveira M. and Castilho-Lopes-de-Costa, J.P. Vroemem, A.J. and Beverini, M Lantero, O.J and Shetty J.K Anastassiadis, S.; Aivasidis, A. and Wandrey, C. Chatterjee, C.; Chatterjee, N. P. and Furtado, E. D. Lantero, Oreste J. and Shetty, J.K.

Novel method for culture of filamentous fungi employed for production of GA Gluconate production by Zymomonas sp. with controlled ethanol production and selective precipitation High yield enzymatic conversion of glucose to GA An enzymatic method that allows high conversion rates of glucose to GA without expensive down stream recovering procedures Process for the production of GA with a strain of Aureobasidium pullulans (de bary) Arnaud Production of gluconate salts Process for the preparation of GA and GA produced thereby

Information based on: http://www.freepatentsonline.com

used, as opposed to the crude forms of grape and banana must (Singh et al. 2005). Despite the progress made so far, studies have yet to meet the demand for a simple carbohydrate substrate that does not carry additional costs for substrate clarification. (4) Inorganic constituentsAfter clarification of fermentable sugars, the alternative substrates may alter total salt composition in the fermentation medium; therefore, balancing the inorganic constituents is important in setting up a medium for GA production. Several studies have been conducted to optimize the balance of nutrients in fermentation media for GA production (Nakamatsu et al. 1975; Mahmoud et al. 1976; Rohr et al. 1983; Ray and Banik 1999; Anastassiadis et al. 2003; Singh et al. 2001b). A lower concentration of nitrate salt leads to an induction of GOX in A. niger, and thus, directly induces GA activity in the fermentation medium (Ray and Banik 1999). A nitrogen concentration greater than 0.25% affects the accumulation of organic acids other than GA (Gupta et al. 1976). Anastassiadis et al. (2003) have suggested that high nitrogen concentration increases activity of the necessary oxidation enzymes by inhibiting significant regulatory enzymes in the glycolytic pathway. In general, phosphate is also required for fungal growth. However, for higher GA accumulation, fungal growth must be regulated. (5) Aeration and modulatorsO2 supply to the microorganism is a key parameter, because O2 is a substrate of the GOX-catalase enzymatic complex and is essentially required for GA formation (Hartmeier and Doppner 1983; Moresi et al. 1991; Trager et al. 1991). In the past, O2 from atmospheric air has been the main source of aeration. When utilizing unconventional carbohydrate sources, the poor solubility of O2 in the aqueous phase could become a bottleneck in the GA fermentation

process. Other than O2, modulators like vegetable oils, H2O2, and starch have marked influence in the fermentation process (Rols et al. 1991; Rols and Goma 1991; Witteveen et al. 1993; Singh and Singh 2006). Adding O2 vector and modulators like vegetable oils to the fermentation medium can enhance the rate of O2 transfer in the medium and accelerate the rate of GA formation by 1520% with 95.8% yield at a conversion rate of 0.720.804 g GA/g rectified grape must (Singh and Singh 2006). The amount of H2O2 is critical, because higher amounts may cause cell death, whereas too little would not release enough O2 because of breakdown of H2O2 by constitutive catalases activity (Fig. 2). Adding H2O2 at a concentration of 1% has been recommended as the best initial fermentation setup with A. niger (Witteveen et al. 1993; Singh and Singh 2006). (6) pH influencepH is one of the most viable factors for continuing the GA fermentation. Fundamental investigations have revealed that organisms such as Aspergillus, Penicillium, and Gluconobacter can effectively grow at a pH range of 2.57.5 with active GA formation at pH values between 5 and 6.5 (Kundu and Das 1984; Roukas and Harvey 1988; Velizarov and Beschkov 1994; Singh et al. 2001b; Znad et al. 2004). A series of enzymes required for GA production (Fig. 2) are only activated at neutral pH, which is usually brought about by adding calcium carbonate and/or sodium carbonate to the fermentation medium as neutralizing agents. Znad et al. (2004) maintained a GA-production rate of 4.583 g/l h1 for 23 h at pH 5.5; the final concentration of GA was 150 g/l in 55 h. Most agro-food by-products have acidic pH (2.53.5; Singh 2000), so it is recommended to start fermentation at the upper pH range (6.07.0; Buzzini et al. 1993; Roukas 2000; Singh et al. 2005; Singh and Singh 2006).

718 Table 6 Major gluconic acid and derivatives manufacturers Manufacturers Alfa Chem Alfa Aesar Ashland Chemical company Aaron Industries, Inc. Biogluconics, Inc. Coyne Chemical Diehl Chemical Glucona America, Inc. Gallard-Schlesinger Industries, Inc. Gleason Chemicals Hydrite Chemical Co. Kelatron Laboratories Miljac Inc. Mihwa Co., Ltd. Omicron Biochemicals, Inc. Pfizer PMP Fermentation Products, Inc. Purac America Inc. Research Organics Inc. Ruger Chemical Co. Inc. US Chemicals, Inc. Westco Chemical, Inc. Wilshire Chemical Co., Inc. Wintersun Chemical
a

Appl Microbiol Biotechnol (2007) 75:713722

Product

tation process for continuous production of GA have been accomplished by extending the frontiers of industrial microbiology through biotechnology approaches. Strain improvement for industrial microorganism (1) MutagenesisA suitable microbial strain is an absolute necessity that can survive over a wide range of complex carbohydrates and higher levels of glucose with little or no toxicity of formed product. Therefore, the attempts to modify the fungal strains for continuous fermentation of GA seem reasonable for a cost effective GA production. To improve GA yield, studies in the past have shown progress towards strain improvement by physical mutagenesis using ultraviolet (UV) irradiation, X-rays (Kundu and Das 1984; Witteveen et al. 1990; Petruccioli et al. 1995; Singh et al. 2001a), and certain chemicals (Markwell et al. 1989; Ray and Banik 1994; Fiedurek and Szczodrak 1995; Singh 2000) to create selective mutants of A. niger and Penicillium sp. with better GA-producing capacity than the parent strain. In an early study, an 11.2% improvement in calcium gluconate production was reported by a mutant of P. funiculosum after treatment with 2-M solution of NaNO3 (Mandal and Chatterjee 1985). In another study, multi-step UV exposure of A. niger resulted in an 87% higher GAproduction level (Singh et al. 2001a). Chemical treatments with nitrous acid and N-methyl-N-nitroN-nitrosoguanidine have also been tried, with only a marginal increase in GA production (Singh 2000). Singh (2006) reported getting a 149% higher level of GOX from mutant A. niger under liquid culture conditions using HCF-treated sugarcane molasses as a cheaper carbohydrate source. The stability of the formed product in several generations of modified microorganism is crucial towards a prolonged survival of any fermentation industry that needs additional efforts to establish further. (2) Genetically engineered microorganismsA strong degeneration in selective mutant strains can occur upon the storage of conidial material; therefore, molecular-biology-based modern genetics and proteins engineering put forth new avenues to create genetically engineered microorganisms (GEMs) that can function as booster biocatalysts. The enzyme GOX involved in bioconversion of D-glucose to GA can be induced significantly using cloned genes in GEMs. A few laboratories have made model organisms for increased GA production by cloning A. niger s gene encoding for GOX (gox A; Swart et al. 1990; Witteveen et al. 1993). These studies indicated that gox A overproduction is independently regulated by

CG, IG, ZG GA Gluconates, CG ZG FG GA GA CA, GA, CG, SG, PG, 6-PG trisodium salt GA GA CG, SG, PG GA GA, CG, SG GA--lactone GA GA, GDL, SG GA, GA--lactone, FG, PG, GDL, SG, ZG CG, 6-PG-Barium slat, trisodium salt, cyclohexylammonium salt, Co G, CG, PG, MG, GA CG, GDL, GA, MGb, MG CG, FG, ZG, GA

Product information provided here is based on the individual manufacturers online catalog. CG, Calcium gluconate; Co G, copper gluconate; FG, ferrous Gluconate; GA, gluconic acid; GDL, glucono--lactone; IG, iron gluconate; MG, manganese Gluconate; MGb , magnesium gluconate; PG, potassium gluconate; 6-PG, 6-phosphogluconate; SG, sodium gluconate; ZG, zinc gluconate

Commercialization of GA production Fermentation industries rely on its commercial uses for product manufacturing unit. Initially, a semipilot scale surface process for GA production was proposed but was held back because of low productivity and limited yield. The first industrial production of GA was established after pilot plant studies in the laboratories of the United States Department of Agriculture. Many processes for industrial GA production have been patented; some from the past decade are summarized in Table 5, and the major manufacturers of GA and its derivatives are listed in Table 6. Recent developments towards a superior fermen-

Appl Microbiol Biotechnol (2007) 75:713722

719

medium compositionmainly carbon and O2 content. Park et al. (2000) cloned and expressed GOX from A. niger in Saccharomyces cerevisiae using a yeast shuttle vector. A gox-encoding gene from P. variabile P16 was isolated and characterized to identify the overexpression with a view to improve the GOX activity in fermentation medium (Pulci et al. 2004). A stable recombinant microbial strain would make it easier and cheaper to convert raw glucose into GA; therefore, studies to construct a recombinant microbial strain for GA fermentation needs further attention.

Fermentation types for GA production Modifications in microbial culture conditions are often useful to test whether production improves under submerged, surface, and other modified fermentation conditions. Based on the mode of O2 supply, two types of culture conditions have been defined for GA fermentation: submerged fermentation (SmF) and solid-state surface fermentation (SSF). (1) Submerged fermentation processRoutine fermentations using conventional microorganisms under submerged conditions led to the development of the SmF process for GA production. Indeed, the benefit of SmF lies in the fact that it can be modified for continuous operation (Fiedurek et al. 1998; Anastassiadis et al. 2003, 2005; Anastassiadis and Rehm 2006a,b). However, it requires high-energy consumption, maintenance, and continuous addition of neutralizing agents, which jeopardize the efficient operation of SmF in fermentation industries. Therefore, a system is needed that can overcome the major drawbacks of SmF. A series of fermentation types, including SmF, surface fermentation (SF), semisolid fermentation (SmSF), and SSF has been evaluated for GA fermentation (Singh et al. 2003). The SSF was found to be more efficient with 94% yield of GA using glucose than any other fermentation types (Singh et al. 2003). (2) Solid-state surface fermentationThe SSF was seeded from SF and designed in shallow pans by developing the fungal mycelium mat on the surface of the medium. Initially, SF was considered to be feasible for continuous GA fermentation process but later rejected because of the poor O2 transfer in liquid phase that do not permit smooth operation for continuous fermentation of GA. Therefore, SF has been modified by employing a perforated solid support to the microorganism using natural substrate as a carbon/energy source in the presence of little or no free-liquid medium to develop SSF process

(Shankaranand et al. 1992; Pandey et al. 1999; Singh et al. 2003). A modified SSF approach featured pseudoimmobilization of A. niger on HCl-pretreated bagasse fibers was observed superior to the SmF and SF processes with 95% GA yield (Singh et al. 2003). The interesting aspect of this method is that bagasse offers multiple downstream benefits as reported by Singh and Singh (2006). Towards utilizing alternative carbohydrate sources under SSF, a two-step process was generated: Spores of A. niger were grown on buckwheat seeds, then converted glucose to 200 g/l GA with a yield of 1.09 g of GA per mass of glucose (Moksia et al. 1996). Roukas (2000) achieved a 63% GA yield from A. niger grown on figs under SSF. SSF has proven to be a cost-effective fermentation process using grape must as a carbohydrate substrate (equivalent to 120 g/l glucose) by A. niger, producing 80 85 g/l GA with a 95.8% yield, at 0.804 g/g substrate conversion rate (Singh and Singh 2006). With grape must as the substrate in SSF, the cellular mat has been successfully reused for five 5-day cycles, generating a 9295.8% GA yield (Singh and Singh 2006). This shows that SSF can be implemented on industrial scale to economize the GA fermentation process.

Immobilized cell fermentation process The biotechnological fermentation of desired compounds generally depends on the density of live microbial cells in the fermentation medium. In addition, continuous fermentation with free cells is adversely affected by cell death and sheared cell lysis, and usually unable to operate under chemostatic mode. Immobilization technology is an attractive and established way to achieve high cell densities to accomplish rapid carbohydrate conversion into organic acids (Vassilev and Vassileva 1992; Vassilev et al. 1993; Sankpal and Kulkarni 2002). GA production has been studied by immobilizing A. niger on glass rings (Heinrich and Rehm 1982), nonwoven fabric material (Sakurai et al. 1989), Ca-alginate (Moresi et al. 1991; Rao and Panda 1994), cross-linking with glutaraldehyde (Rao and Panda 1994), and polyurethane foam (PUF; Vassilev et al. 1993), including flocculation with the polyelectrolytes and covalent binding to glycidyl co-polymers that have been guarded by patents. Sankpal et al. (1999) reported a capillary-based vertical fabric support for A. niger that can run for a period of 61 days with 120140 g/l emerging GA. Later, Sankpal and Kulkarni (2002) found that the optimum biomass for efficient GA bioconversion on a porous cellulose support was 0.234 mg/cm2. Yeast-like strain A. pullulans was immobilized on porous-sintered glass and applied in a fluidized bed reactor under defined fermenta-

720

Appl Microbiol Biotechnol (2007) 75:713722

tion medium using 450 g/l glucose; this produced 315 g/l GA in a continuous chemostat culture after 21 h (Anastassiadis and Rehm 2006b). To develop a semicontinuous GA-production method utilizing unconventional carbohydrate sources, Vassilev et al. (1993) studied whole-cell immobilization of A. niger on PUF in hydrol-based medium; the highest GA concentration achieved was about 143 g/l. Whole-cell immobilization of A. niger in calcium alginate using potassiumferrocyanide-treated cane molasses as a substrate had a better product yield (0.40 g GA/g total reducing sugar supplied) than the cross-linking with glutaraldehyde (Rao and Panda 1994). In a detailed study, Singh (2000) tested a variety of alternative carbohydrate sources such as sugarcane molasses, grape must, and banana must for semicontinuous production of GA using PUS- and calcium-alginateimmobilized whole cells of A. niger. Semicontinuous GA production with 98% yield was achieved using grape must with PUS-immobilized A. niger (Singh 2000). PUFsupported A. niger produced 92 g of GA from 1 l deproteinized whey supplemented with lactose and glucose than 69 g of GA from free mycelia (Mukhopadhyay et al. 2005). Much work remains to be done concerning the use of PUS- and Ca-alginate-matrices with unconventional carbohydrate sources for continuous GA production.

rectified grape must (Singh 2000). Some loss of GA was observed during decolorization, yielding free GA with 89% of the total recovery. Thus, 120 g/l equivalent glucose from rectified grape must results in 94 g of pure GA from the fermented broth after 10 days of SSF (Singh 2000). Therefore, the classical methods of separating purified GA would be an advantage to the fermentation industries, with no extra expenditure in downstream processing.

Conclusion GA production is a simple oxidation process that can be carried out by multiple modes of reaction; microbial fermentation has become a viable mode for commercial production. To make the traditional fermentation process more economical, researchers have evaluated cheaper carbohydrate substrates and made innovative modifications to the process. Among agro-food by-products, sugarcane molasses and grape must can be used as alternative carbohydrate substrates to produce GA under SSF on an industrial scale, which will eventually pave the way to a cheaper cell-free enzymatic bioprocess for GA production.
Acknowledgements Thanks to Dr. Amal Das of the University of North Carolina for useful discussions. Technical support rendered by Rashmi Singh for preparing this manuscript is gratefully acknowledged. We also thank the reviewers and the editorial team for their insightful suggestions regarding the review content.

Product recovery Obtaining the desired product in pure form after fermentation with alternative carbohydrate sources may be one of the tedious tasks for fermentation industries. The presence of neutralizing agents such as calcium and/or sodium ions is an advantage in recovering purified GA. The downstream processing of GA is generally similar for the fermentation processes employed by fungal and bacterial species after the mycelium is separated from the fermentation broth. Free GA can be recovered from calcium gluconate either by classical calcium sulphate precipitation or by passing the solution through a column containing a strong acid-cation exchanger to absorb the calcium ions (Milson and Meers 1985). Purified Glucono--lactone crystals can be obtained from supersaturated solutions of GA at 3070C. Blom et al. (1952) recovered 45% solids of sodium gluconate by simply concentrating the filtered fermentation broth, then adding sodium hydroxide to pH 7.5 and drum drying. A variant method of recovering GA from a clarified and decolorized broth containing sodium gluconate has been patented whereby the solution is passed through a strong anion exchanger such as the Amberlite IRS400, which retains the GA in the column. The classical GA-recovery process was found particularly suitable for use with impure liquors derived from

References
Anastassiadis S, Rehm HJ (2006a) Continuous gluconic acid production by the yeast-like Aureobasidium pullulans in a cascading operation of two bioreactors. Appl Microbiol Biotechnol 73:541 548 Anastassiadis S, Rehm HJ (2006b) Continuous gluconic acid production by Aureobasidium pullulans with and without biomass retention. Electron J Biotechnol 9:494504 Anastassiadis S, Aivasidis A, Wandrey C (2003) Continuous gluconic acid production by isolated yeast-like mould strains of Aureobasidium pullulans. Appl Microbiol Biotechnol 61:110117 Anastassiadis S, Aivasidis A, Wandrey C, Rehm HJ (2005) Process optimization of continuous gluconic acid fermentation by isolated yeast-like strains of Aureobasidium pullulans. Biotechnol Bioeng 91:494501 Blom RH, Pfeifer VF, Moyer AJ, Traufler DH, Conway HF, Crocker CK, Farison RE, Hannibal DV (1952) Sodium gluconate production. Ind Eng Chem 44:435440 Business Communication Co. (BCC) (2004) GA-103R-World markets for fermentation ingredients. Norwalk, CT, USA Buzzini P, Gabbbetti M, Rossi J, Ribaldi M (1993) Utilization of grape must and concentration rectified grape must to produce gluconic acid by Aspergillus niger, in batch fermentation. Biotechnol Lett 15:151156 Clark DS, Ito K, Horitsu H (1966) Effect of manganese and other heavy metals on submerged citric acid fermentation of molasses. Biotechnol Bioeng 8:465471

Appl Microbiol Biotechnol (2007) 75:713722 Fiedurek J, Szczodrak J (1995) Glucose oxidase biosynthesis in relation to biochemical mutations in Aspergillus niger. Acta Biotechnol 15:107115 Fiedurek J, Gromada A, Pielecki J (1998) Simultaneous production of catalase, glucose oxidase and gluconic acid by Aspergillus niger mutant. Acta Microbiol Pol 47:355364 Fischer K, Bipp HP (2005) Generation of organic acids and monosaccharides by hydrolytic and oxidative transformation of food processing residues. Bioresour Technol 96:831842 Gupta JK, Heding LG, Jargensen OB (1976) Effect of sugars, pH and ammonium nitrate on formation of citric acid by Aspergillus niger. Acta Microbiol Acad Sci Hung 23:6367 Hartmeier W, Doppner T (1983) Preparation and properties of mycelium bound glucose oxidase co-immobilized with excess catalase. Biotechnol Lett 5:743748 Heinrich M, Rehm HJ (1982) Formation of gluconic acid at low pHvalues by free and immobilized Aspergillus niger cells during citric acid fermentation. Eur J Appl Microbiol Biotechnol 15:8892 Herrick HT, May OE (1928) The production of gluconic acid by the Penicillium luteum- purpurogenum group. J Biol Chem 77:185195 Hustede H, Haberstroch HJ, Schinzig E (1989) Gluconic acid. In: ULLMANNS encyclopedia of industrial chemistry, vol A 12. VCH-Weinheim, pp 449456 Ikeda Y, Park EY, Okuda N (2006) Bioconversion of waste office paper to gluconic acid in a turbine blade reactor by the filamentous fungus Aspergillus niger. Bioresour Technol 97:10301035 Klein J, Rosenberg M, Marko J, Dolgo O, Krolk M, Kritofkov L (2002) Biotransformation of glucose to gluconic acid by Aspergillus niger-study of mass transfer in an airlift bioreactor. Biochem Eng J 10:197205 Kundu P, Das A (1984) Utilization of cheap carbohydrate sources for production of calcium gluconate by Penicillium funiculosum mutant MN 238. Indian J Exp Biol 22:279281 Kundu S, Panda T, Majumdar SK, Guha B, Bandyopadhyay KK (1984) Pretreatment of Indian cane molasses for increased production of citric acid. Biotechnol Bioeng 26:11141121 Mahmoud SA, El-Sawy M, Nour El-Din Ibrahim OO (1976) Studies on some nutritional factors influencing the production of gluconic acid. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt 1 Suppl 131:361374 Mandal SK, Chatterjee SP (1985) Improved production of calcium gluconate by mutants of Penicillium funiculosum. Curr Sci 54:149 Markwell J, Frakes LG, Brott EC, Osterman J, Wagner FW (1989) Aspergillus niger mutants with increased glucose oxidase production. Appl Microbiol Biotechnol 30:166169 Milson PE, Meers JL (1985) Gluconic acid, itaconic acid. In: Blanch HW, Drew S, Wang DIC (eds) Comprehensive biotechnology, vol. 3. Pergamon, Oxford, pp 681700 Moksia J, Larroche C, Gros JB (1996) Gluconate production by spores of Aspergillus niger. Biotechnol Lett 18:10251030 Moresi M, Parente E, Mazzatura A (1991) Effect of dissolved oxygen concentration on repeated production of gluconic acid by immobilized mycelia of Aspergillus niger. Appl Microbiol Biotechnol 36:320323 Mukhopadhyay R, Chatterjee S, Chatterjee BP, Banerjee PC, Guha AK (2005) Production of gluconic acid from whey by free and immobilized Aspergillus niger. Int Dairy J 15:299303 Nakamatsu T, Akamatsu T, Miyajima R, Shio I (1975) Microbial production of glucose oxidase. Agric Biol Chem 39:18031811 Pandey A, Selvakumar P, Soccol CR, Nigam P (1999) Solid state fermentation for the production of industrial enzymes. Curr Sci 77:149162

721 Park EH, Shin YM, Lim YY, Kwon TH, Kim DH, Yang MS (2000) Expression of glucose oxidase by using recombinant yeast. J Biotechnol 81:3544 Petruccioli M, Piccioni P, Dederici F, Polsineli M (1995) Glucose oxidase overproducing mutants of Penicillium variable (P16). FEMS Microbiol Lett 128:107112 Pulci V, DOvidio R, Petruccioli M, Federici F (2004) The glucose oxidase of Penicillium variabile P16: gene cloning, sequencing and expression. Lett Appl Microbiol 38:233238 Ramachandran S, Fontanille P, Pandey A, Larroche C (2006) Gluconic acid: properties, applications and microbial production, a review. Food Technol Biotechnol 44:185195 Rao DS, Panda T (1994) Comparative analysis of different whole cell immobilized Aspergillus niger catalysts for gluconic acid fermentation using pretreated cane molasses. Bioprocess Biosyst Eng 11:209212 Rao D, Panda T, Rao DS (1994) Critical analysis of the effect of metal ions on gluconic acid production by Aspergillus niger using a treated cane molasses. Bioprocess Biosyst Eng 10:99107 Ray S, Banik AK (1994) Development of a mutant strain of Aspergillus niger and optimization of some physical factors for improved calcium gluconate production. Indian J Exp Biol 32:865868 Ray S, Banik AK (1999) Effect of ammonium and nitrate ratio on glucose oxidase activity during gluconic acid fermentation by a mutant strain of Aspergillus niger. Indian J Exp Biol 37:391395 Roehr M, Kubicek CP, Kominek J (1996) Gluconic acid. In: Rehm HJ, Reed G (eds). Biotechnology, product of Primary Metabolism, vol. 6. Verlag Chemie, Weinheim, pp. 347362 Rohr M, Kubicek CP, Kominek J (1983) Citric acid, gluconic acid. In: Rehm HJ, Reed G (eds). Biotechnology, vol. 3. Verlag Chemie, Weinheim, pp 420465 Rols JL, Goma G (1991) Enhanced oxygen transfer rates in fermentation using soybean oil-in-water dispersions. Biotechnol Lett 13:712 Rols JL, Condorect JS, Fonade C, Goma G (1991) Modeling of oxygen transfer in water emulsified organic liquids. Chem Eng Sci 46:18691873 Roukas T (2000) Citric acid gluconic acid production from fig by Aspergillus niger using solid-state fermentation. J Ind Microbiol Biotech 25:298304 Roukas T, Harvey L (1988) The effect of pH on production of citric acid and gluconic acid from beet molasses using continuous culture. Biotechnol Lett 10:295300 Sakurai H, Lee HW, Sato S, Mukataka S, Takahashi J (1989) Gluconic acid production at high concentration by Aspergillus niger immobilized on a nonwoven fabric. J Ferment Bioeng 67:404408 Sankpal NV, Kulkarni BD (2002) Optimization of fermentation conditions for gluconic acid production using Aspergillus niger immobilized on cellulose microfibrils. Process Biochem 37:13431350 Sankpal NV, Joshi AP, Sutar II, Kulkarni BD (1999) Continuous production of gluconic acid by Aspergillus niger immobilized on a cellulosic support: study of low pH fermentative behaviour of Aspergillus niger. Process Biochem 35:317325 Shankaranand VS, Ramesh MV, Lonsane BK (1992) Idiosyncrasies of solid state fermentation systems in the biosynthesis of metabolites by some bacterial and fungal cultures. Process Biochem 27:3336 Silveira MM, Wisbeck E, Lemmel C, Erzinger G, da Costa JP, Bertasso M, Jonas R. (1999) Bioconversion of glucose and fructose to sorbitol and gluconic acid by untreated cells of Zymomonas mobilis. J Biotechnol 75:99103 Singh OV (2000) Bioconversion of agro-food by-products to gluconic acid by Aspergillus niger. Ph.D. thesis, Indian Institute of Technology, Roorkee, India (Formerly University of Roorkee)

722 Singh OV (2006) Mutagenesis and analysis of mold Aspergillus niger for extracellular glucose oxidase production using sugarcane molasses. Appl Biochem Biotechnol 135:4358 Singh OV, Singh RP (2006) Bioconversion of grape must into modulated gluconic acid production by Aspergillus niger ORS4.410. J Appl Microbiol 100:11141122 Singh OV, Sharma A, Singh RP (2001a) Mutagenesis and production of gluconic acid by Aspergillus niger mutant ORS-4.410 in submerged and solid state surface cultivation. Indian J Exp Biol 39:691696 Singh OV, Sharma A, Singh RP (2001b) Optimization of fermentation conditions for gluconic acid production by mutant Aspergillus niger. Indian J Exp Biol 39:11361143 Singh OV, Jain RK, Singh RP (2003) Gluconic acid production under varying fermentation conditions by Aspergillus niger. J Chem Technol Biotechnol 78:208212 Singh OV, Kapur N, Singh RP (2005) Evaluation of agro-food byproducts for gluconic acid fermentation by Aspergillus niger ORS-4.410. World J Microbiol Biotechnol 21:519524 Swart K, van de Vondervoort PJ, Witteveen CF, Visser J (1990) Genetic localization of a series of genes affecting glucose oxidase levels in Aspergillus niger. Curr Genet 18:435439

Appl Microbiol Biotechnol (2007) 75:713722 Trager M, Quzi GN, Onken U, Chopra CL (1991) Contribution of endo and extracellular glucose oxidase to gluconic acid production at increased dissolved oxygen concentration. J Chem Technol Biotechnol 50:111 Vassilev N, Vassileva M (1992) Production of organic acids by immobilized filamentous fungi. Mycol Res 96:563570 Vassilev N, Vassileva MC, Spassova DI (1993) Production of gluconic acid by Aspergillus niger immobilized on polyurethane foam. Appl Microbiol Biotechnol 39:285288 Velizarov S, Beschkov V (1994) Production of free gluconic acid by cells of Gluconobacter oxydans. Biotechnol Lett 16:715720 Witteveen CFB, Vondervoot VDP, Swart K, Visser J (1990) Glucose oxidase overproducing and negative mutants of Aspergillus niger. Appl Microbiol Biotechnol 33:683686 Witteveen CFB, Vondervoort VDP, Van Den Broeck HC, Van Engelenburg FAC, De Graff LH, Hillebrand MHBC, Schaap PJ, Visser J (1993) The induction of glucose oxidase, catalase and lactonase in Aspergillus niger. Curr Genet 24:408406 Znad H, Markos J, Bales V (2004) Production of gluconic acid from glucose by Aspergillus niger growth and non-growth conditions. Process Biochem 39:13411345