biotechnological production of gluconic acid future implecations

8/13/2019 Biotechnological production of gluconic acid future implecations

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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 carbonicacid regarded as a bulk chemical in the food, feed, beverage,textile, pharmaceutical, and construction industries. Thefavored production process is submerged fermentation by Aspergillus niger utilizing glucose as a major carbohydratesource, which accompanied product yield of 98%. Howev-er, use of GA and its derivatives is currently restricted because of high prices: about US$ 1.20 8.50/kg. Advance-ments in biotechnology such as screening of microorgan-isms, immobilization techniques, and modifications infermentation process for continuous fermentation, includinggenetic engineering programmes, could lead to cost-effective production of GA. Among alternative carbohy-drate sources, sugarcane molasses, grape must show highest GA yield of 95.8%, and banana must may assist reducingthe overall cost of GA production. These methodologieswould open new markets and increase applications of GA.

Keywords Gluconic acid . Microbial fermentation . Glucoseoxidase . Alternative carbohydrate sources . Biotechnology .System biology

Introduction

Because of rising interest in sustainable development, there isextensive demand in the construction, chemical, and pharma-ceutical industries for an absolute polyhydroxycarboxylicacid that has both reactive hydroxyl and carboxyl groups.Gluconic acid (GA, penta-hydroxycaproic acid; Fig. 1) is anaturally occurring polyhydroxycarboxylic acid commonlyfound in humans and other organisms. GA has beenmanufactured so far by discontinuous, intermittent batchand fed batch processes. The low selectivity in chemicalsynthesis of GA is uneconomical for industrial purposes(Hustede et al. 1989). Hence, the microbial conversion of glucose into GA in submerged fermentation process employ-ing fungal species like A. niger and Penicillium and bacterialspecies such as Pseudomonas, Acetobacter , and Glucono-bacter , etc. has been reviewed in the past (Milson and Meers1985 , 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 require-ments in fermentation contribute to the high price of GA andits derivatives (Table 1). However, its huge market con-sumption (Table 2) has spurred interest in the development of an effective and economical system for GA production.

Among its other outstanding properties, GA has anexcellent chelating capacity in alkaline solutions. GA has been extensively used in the cleaning and constructionindustries as an additive to increase cement resistance andstability under extreme climatic conditions (Milson andMeers 1985; Roehr et al. 1996; Ramachandran et al. 2006).Thus, the enormous application-dependent growth of GAenhanced the total market value: about US$333 million(Table 3; Business Communication Co. 2004).

Appl Microbiol Biotechnol (2007) 75:713 722DOI 10.1007/s00253-007-0851-x

Authors contributions OVS and RK are the sole contributors of thisoriginal review article. This review is based upon the publishedresearch 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, USAe-mail: [email protected]: [email protected]

R. Kumar Division of Radiation Biology,Institute of Nuclear Medicine and Allied Sciences, New Delhi 110 054, India


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The surplus demand is about 87,000 tonnes (Table 2;Business Communication Co. 2004), and an estimatedhigher cost of US$ 1.20 8.50/kg of GA and its derivatives(Table 1) can be conquered by exploring unconventionalcarbohydrate sources such as agro-food by-products andmodifications to the fermentation process. A wide variety of cheaper carbohydrate sources including sugarcane molasses, beet molasses, grape must, banana must, and paper wastehave been proposed as substrates for GA production with85 95% yield (Kundu and Das 1984; Roukas and Harvey1988 ; Buzzini et al. 1993; Rao et al. 1994; Rao and Panda1994 ; Singh et al. 2003, 2005; Ikeda et al. 2006; Singh andSingh 2006). The key question is whether it is possible toexploit the availability of alternative carbohydrate sources todevelop 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 onGA production using glucose, the present article deals withthe crucial parameters for developing an indigenoustechnology for GA fermentation by exploring a variety of cheaper alternative carbohydrate sources. A critical set of fermentation processes suitable for continuous GA fermen-tation is also discussed.

Microorganisms: asset in GA fermentation!

Microorganisms have excelled at producing primary andsecondary metabolites from a variety of raw carbohydratessince billions of years, and one of them was alcoholicfermentation. In current vogue, the results of studying giant microbial libraries for microbial conversion of cheaper carbohydrates into value-added products can serve as a rawmaterial for GA fermentation.

In fungi such as A. niger , GA is a product of simpledehydrogenation catalyzed by glucose oxidase (GOX) fromD-glucose (Fig. 2). This oxidation reaction of an aldehydegroup forms a carboxylic group resulting in glucono- -lactone and H 2 O2 , which further hydrolyzes into GA

spontaneously or via a lactone-hydrolyzing enzyme decom- posing H 2 O2 into water and oxygen (Fig. 2). Besides A.niger , other species of the genera such as Penicillium ,Gliocadium , Scopulariopsis , and Gonatobotrys have beentested for GA production and reviewed by Milson andMeers (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 , andScopulariopsis participate in GA production with a specific pathway by oxidizing glucose into GA with glucosedehydrogenase (Fig. 2); this process has been reviewed byMilson and Meers ( 1985) and Ramachandran et al. ( 2006).

In the past, a variety of microorganisms have beenexplored that can utilize the alternative carbohydratesources (such as hydrol, corn starch, grape must, bananamust, fig, cheese whey, food processing residues, andsaccharified solution of waste paper) with an 80 95% yieldof GA in submerged and solid-state surface fermentation(Roukas and Harvey 1988; Buzzini et al. 1993; Roukas2000; Mukhopadhyay et al. 2005; Singh et al. 2005; Ikedaet al. 2006; Singh and Singh 2006). A. niger was grown inconcentrated, rectified grape must with 67.43 g/l GA and ayield of 96% (converted glucose; Buzzini et al. 1993), and96.52 g/l with a 95% yield of GA (Singh and Singh 2006). A. niger ATCC 10577 was employed on fig fruit for GAand citric acid production with 490 g GA/kg dry fig

Fig. 1 Gluconic acid formulaand some physical properties

Table 1 Market value of gluconic acid and derivatives based uponmajor industrial applicability (Source: Business Communication Co.,Inc. 2004)

Major product andsalts

Applicability Average cost ($/kg)

Gluconic acid Cement industries 1.20Sodium gluconate Cement industries 2.00Pure sodium gluconate Food industry 3.00Calcium gluconate and -lactone

Food and pharamaceuticalindustries

8.50

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(Roukas 2000). Another strain of A. niger NCIM 548 wasgrown on deproteinised whey as a nutritive medium producing 92 g GA/l whey (Mukhopadhyay et al. 2005).The microorganism A. niger IAM 2094, which consumesglucose 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 processusing A. pullulans offers a new opportunity for commercialGA production, with 504 g/l GA in fed batch and 400 g/l inchemostate operations produced by free-growing cells witha maximum of 96.7% yield (Anastassiadis et al. 2003). Insupport of this study, it is known that A. niger is difficult tohandle 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 highglucose concentrations. A. pullulans integrates the advan-tages of both fungal and bacterial systems, utilizing ahigher glucose concentration in continuous operation to produce 350 433 g/l GA (Anastassiadis and Rehm 2006b).However, the growth and GA-production efficiency of thismicroorganism over a wide range of complex carbohydratesis still unclear.

Evaluation of GA fermentation process

(1) Fermentation medium Each microorganism has itsown adaptability on a variety of fermentation media.However, several essential nutrients are required for any fermentation reaction. A variety of constituentshave been defined in the past for GA fermentationmedia 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 systematicstudy of the influence of medium composition on GA production by P. luteum purpunogenum group organ-isms. A critical analysis of salt concentration can

maximize GA production and cut down on contami-nating side products generated from A. niger duringthe transformation process (Moksia et al. 1996). Inaddition to minor elements, media containing traces of magnesium, potassium, and phosphate salts are most suitable for A. niger with 80 85 g/l GA productionwhile exploring grape must as a carbohydrate source(Singh and Singh 2006).

(2) Carbon sources The smooth operation of any fer-mentation industry largely depends on the cost of manufacture, and this in turn depends on the selectionof raw materials. Glucose at a concentration of 10

15%, either in the form of glucose monohydratecrystals or fructose/dextrose syrup, has long been proposed as the main carbon source for GA produc-tion, with a 90 95% yield (Silveira et al. 1999; Singhet al. 2001a, b; Klein et al. 2002; Znad et al. 2004).However, because microorganisms can be grown on awide variety of carbohydrate sources, the agro-food by-products can be considered as an economicalsource of carbohydrates for GA fermentation. Amongthe major agro-food by-products, sugarcane molassesand grape must have high sugar content and can beused for the production of alcohol, which makes themsuitable candidates for the GA fermentation process.

The alternative carbohydrate sources with the potentialto make the GA fermentation process more economical arehydrol, corn starch, can molasses, grape must, banana must,food processing residues, figs, cheese whey, beet molasses,and saccharified solution of waste paper (Kundu and Das1984 ; Roukas and Harvey 1988 ; Buzzini et al. 1993 ; Raoand 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 ). Inanother attempt using fig fruit, Roukas ( 2000 ) obtained490 g GA/kg dry fig a 63% yield and found that addingmodulators such as 6% methanol into substrate enhancesthe levels of GA (685 g GA/kg dry fig). A by-product of dairy industry, deproteinised whey containing 9.5% lactose

Table 2 World wide consumption of gluconic acid based on major industrial applications (Source: Business Communication Co., Inc.2004 )

Usage Quantity(in tonnes)

Total consumption(%)

Pharmaceutical industries 8,000 9.2Food industries 30,000 34.5Construction industries 40,000 46.0Other usage 9,000 10.3Total 87,000 100.0

Table 3 Major application-based global market expenditure in past and future assumption of gluconic acid (Source: Business Communi-cation Co., Inc. 2004)

Application 2004(US$ million)

2009(US$ millions)

AAGR%2004 2009

Construction 60 76 4.8Food 150 153 0.4Pharmaceuticals 63 51 5.6Others 55 31 10.8Total 333 311 1.4

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used, as opposed to the crude forms of grape and bananamust (Singh et al. 2005 ). Despite the progress made so far,studies have yet to meet the demand for a simplecarbohydrate substrate that does not carry additional costsfor substrate clarification.(4) Inorganic constituents After clarification of ferment-

able sugars, the alternative substrates may alter totalsalt composition in the fermentation medium; there-fore, balancing the inorganic constituents is important in setting up a medium for GA production. Severalstudies have been conducted to optimize the balanceof 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 fermen-tation medium (Ray and Banik 1999). A nitrogenconcentration greater than 0.25% affects the accumu-lation of organic acids other than GA (Gupta et al.1976). Anastassiadis et al. ( 2003) have suggested that high nitrogen concentration increases activity of thenecessary oxidation enzymes by inhibiting significant regulatory enzymes in the glycolytic pathway. Ingeneral, phosphate is also required for fungal growth.However, for higher GA accumulation, fungal growthmust be regulated.

(5) Aeration and modulators O2 supply to the microor-ganism is a key parameter, because O 2 is a substrate of the GOX-catalase enzymatic complex and is essentiallyrequired 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 carbohydratesources, the poor solubility of O 2 in the aqueous phasecould become a bottleneck in the GA fermentation

process. Other than O 2 , modulators like vegetable oils,H2 O2 , and starch have marked influence in thefermentation process (Rols et al. 1991; Rols and Goma1991; Witteveen et al. 1993; Singh and Singh 2006).Adding O 2 vector and modulators like vegetable oils tothe fermentation medium can enhance the rate of O 2transfer in the medium and accelerate the rate of GAformation by 15 20% with 95.8% yield at a conversionrate of 0.72 0.804 g GA/g rectified grape must (Singhand Singh 2006). The amount of H 2 O2 is critical, because higher amounts may cause cell death, whereastoo little would not release enough O 2 because of breakdown of H 2 O2 by constitutive catalases activity(Fig. 2). Adding H 2 O2 at a concentration of 1% has beenrecommended as the best initial fermentation setup with A. niger (Witteveen et al. 1993; Singh and Singh 2006).

(6) pH influence pH is one of the most viable factors for continuing the GA fermentation. Fundamental inves-tigations have revealed that organisms such as Asper- gillus , Penicillium , and Gluconobacter can effectivelygrow at a pH range of 2.5 7.5 with active GAformation at pH values between 5 and 6.5 (Kunduand Das 1984; Roukas and Harvey 1988; Velizarovand Beschkov 1994; Singh et al. 2001b; Znad et al.2004). A series of enzymes required for GA produc-tion (Fig. 2) are only activated at neutral pH, which isusually brought about by adding calcium carbonateand/or sodium carbonate to the fermentation mediumas neutralizing agents. Znad et al. ( 2004) maintained aGA-production rate of 4.583 g/l h

1 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 (2.5 3.5;Singh 2000), so it is recommended to start fermenta-tion at the upper pH range (6.0 7.0; Buzzini et al.1993; Roukas 2000; Singh et al. 2005; Singh andSingh 2006).

Table 5 Major US patents in last 10 years for gluconic acid/gluconate production processes a

Patent number

Patent year

Author Patent process

EP649899 1995 Kiyoshi, A and Yosuo , M Novel method for culture of filamentous fungi employed for production of GA

BR9403981 1996 Jonas, R.H.H.H.; Moura-de-Silveira M. andCastilho-Lopes-de-Costa, J.P.

Gluconate production by Zymomonas sp. with controlled ethanol production and selective precipitation

WO9635800 1996 Vroemem, A.J. and Beverini, M High yield enzymatic conversion of glucose to GAWO9724454 1997 Lantero, O.J and Shetty J.K An enzymatic method that allows high conversion rates of glucose

to GA without expensive down stream recovering procedures5962286 1999 Anastassiadis, S.; Aivasidis, A.

and Wandrey, C.Process for the production of GA with a strain of Aureobasidium pullulans (de bary) Arnaud

6828130 2004 Chatterjee, C.; Chatterjee, N. P.and Furtado, E. D.

Production of gluconate salts

6942997 2005 Lantero, Oreste J. and Shetty, J.K. Process for the preparation of GA and GA produced thereby

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

http://www.freepatentsonline.com/http://www.freepatentsonline.com/


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Commercialization of GA production

Fermentation industries rely on its commercial uses for product manufacturing unit. Initially, a semipilot scalesurface process for GA production was proposed but washeld 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 StatesDepartment of Agriculture. Many processes for industrialGA 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 inTable 6. Recent developments towards a superior fermen-

tation process for continuous production of GA have beenaccomplished by extending the frontiers of industrialmicrobiology through biotechnology approaches.

Strain improvement for industrial microorganism

(1) Mutagenesis A suitable microbial strain is anabsolute necessity that can survive over a wide rangeof complex carbohydrates and higher levels of glucosewith little or no toxicity of formed product. Therefore,the attempts to modify the fungal strains for contin-uous fermentation of GA seem reasonable for a cost effective GA production. To improve GA yield,studies in the past have shown progress towards strainimprovement by physical mutagenesis using ultravio-let (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-producingcapacity than the parent strain. In an early study, an11.2% improvement in calcium gluconate productionwas reported by a mutant of P. funiculosum after treatment with 2-M solution of NaNO 3 (Mandal andChatterjee 1985). In another study, multi-step UVexposure of A. niger resulted in an 87% higher GA- production level (Singh et al. 2001a). Chemicaltreatments with nitrous acid and N -methyl- N -nitro- N -nitrosoguanidine have also been tried, with only amarginal increase in GA production (Singh 2000).Singh (2006) reported getting a 149% higher level of GOX from mutant A. niger under liquid cultureconditions using HCF-treated sugarcane molasses asa cheaper carbohydrate source. The stability of theformed product in several generations of modifiedmicroorganism is crucial towards a prolonged survivalof any fermentation industry that needs additionalefforts to establish further.

(2) Genetically engineered microorganisms A strong degeneration in selective mutant strains can occur upon the storage of conidial material; therefore,molecular-biology-based modern genetics and proteinsengineering put forth new avenues to create geneti-cally engineered microorganisms (GEMs) that canfunction as booster biocatalysts . The enzyme GOXinvolved in bioconversion of D-glucose to GA can beinduced significantly using cloned genes in GEMs. Afew laboratories have made model organisms for increased GA production by cloning A. niger s geneencoding for GOX ( gox A; Swart e t a l. 1990;Witteveen et al. 1993). These studies indicated that gox A overproduction is independently regulated by

Table 6 Major gluconic acid and derivatives manufacturers

Manufacturers Product a

Alfa Chem CG, IG, ZGAlfa Aesar GAAshland Chemicalcompany

Gluconates, CG

Aaron Industries, Inc. ZGBiogluconics, Inc. FGCoyne Chemical GADiehl Chemical GAGlucona America, Inc. CA, GA,Gallard-Schlesinger Industries, Inc.

CG, SG, PG, 6-PG trisodium salt

Gleason Chemicals GAHydrite Chemical Co. GAKelatron Laboratories CG, SG, PGMiljac Inc. GAMihwa Co., Ltd. GA, CG, SGOmicronBiochemicals, Inc.

GA- -lactone

Pfizer GAPMP FermentationProducts, Inc.

GA, GDL, SG

Purac America Inc. GA, GA- -lactone, FG, PG, GDL, SG, ZGResearch Organics Inc. CG, 6-PG-Barium slat, trisodium salt,

cyclohexylammonium salt,Ruger Chemical Co.Inc.

Co G, CG, PG, MG,

US Chemicals, Inc. GAWestco Chemical, Inc. CG, GDL, GA, MG b , MGWilshire ChemicalCo., Inc.

CG, FG, ZG,

Wintersun Chemical GA

a Product information provided here is based on the individualmanufacturers online catalog.

CG , Calcium gluconate; Co G , copper gluconate; FG ,ferrous Gluconate; GA, gluconic acid; GDL , glucono- -lactone; IG ,iron gluconate; MG , manganese Gluconate; MG b , magnesiumgluconate; PG , potassium gluconate; 6-PG , 6-phosphogluconate; SG ,sodium gluconate; ZG , zinc gluconate



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medium composition mainly carbon and O 2 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. variabileP16 was isolated and characterized to identify theoverexpression with a view to improve the GOXactivity in fermentation medium (Pulci et al. 2004). Astable recombinant microbial strain would make it easier and cheaper to convert raw glucose into GA;therefore, studies to construct a recombinant microbialstrain for GA fermentation needs further attention.

Fermentation types for GA production

Modifications in microbial culture conditions are oftenuseful to test whether production improves under sub-merged, surface, and other modified fermentation con-

ditions. Based on the mode of O 2 supply, two types of culture conditions have been defined for GA fermentation:submerged fermentation (SmF) and solid-state surfacefermentation (SSF).

(1) Submerged fermentation process Routine fermenta-tions using conventional microorganisms under sub-merged conditions led to the development of the SmF process for GA production. Indeed, the benefit of SmFlies in the fact that it can be modified for continuousoperation (Fiedurek et al. 1998; Anastassiadis et al.2003 , 2005; Anastassiadis and Rehm 2006a, b). How-ever, it requires high-energy consumption, mainte-nance, and continuous addition of neutralizing agents,which jeopardize the efficient operation of SmF infermentation industries. Therefore, a system is neededthat can overcome the major drawbacks of SmF. Aseries of fermentation types, including SmF, surfacefermentation (SF), semisolid fermentation (SmSF),and SSF has been evaluated for GA fermentation(Singh et al. 2003). The SSF was found to be moreefficient with 94% yield of GA using glucose than anyother fermentation types (Singh et al. 2003).

(2) Solid-state surface fermentation The SSF was seed-ed from SF and designed in shallow pans bydeveloping the fungal mycelium mat on the surfaceof the medium. Initially, SF was considered to befeasible for continuous GA fermentation process but later rejected because of the poor O 2 transfer in liquid phase that do not permit smooth operation for continuous fermentation of GA. Therefore, SF has been modif ied by employing a perforated solidsupport to the microorganism using natural substrateas 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; Singhet al. 2003). A modified SSF approach featured pseudoimmobilization of A. niger on HCl-pretreated bagasse fibers was observed superior to the SmF andSF processes with 95% GA yield (Singh et al. 2003).The interesting aspect of this method is that bagasseoffers multiple downstream benefits as reported bySingh and Singh ( 2006). Towards utilizing alternativecarbohydrate sources under SSF, a two-step processwas generated: Spores of A. niger were grown on buckwheat seeds, then converted glucose to 200 g/l GAwith 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. SSFhas proven to be a cost-effective fermentation processusing grape must as a carbohydrate substrate (equiva-lent to 120 g/l glucose) by A. niger , producing 80

85 g/l GA with a 95.8% yield, at 0.804 g/g substrateconversion rate (Singh and Singh 2006). With grapemust as the substrate in SSF, the cellular mat has beensuccessfully reused for five 5-day cycles, generating a92 95.8% GA yield (Singh and Singh 2006). Thisshows that SSF can be implemented on industrial scaleto economize the GA fermentation process.

Immobilized cell fermentation process

The biotechnological fermentation of desired compoundsgenerally depends on the density of live microbial cells inthe fermentation medium. In addition, continuous fermen-tation with free cells is adversely affected by cell death andsheared cell lysis, and usually unable to operate under chemostatic mode. Immobilization technology is an attrac-tive and established way to achieve high cell densities toaccomplish rapid carbohydrate conversion into organicacids (Vassilev and Vassileva 1992; Vassilev et al. 1993;Sankpal and Kulkarni 2002). GA production has beenstudied by immobilizing A. niger on glass rings (Heinrichand Rehm 1982), nonwoven fabric material (Sakurai et al.1989), Ca-alginate (Moresi et al. 1991; Rao and Panda1994), cross-linking with glutaraldehyde (Rao and Panda1994), and polyurethane foam (PUF; Vassilev et al. 1993),including flocculation with the polyelectrolytes and cova-lent binding to glycidyl co-polymers that have beenguarded by patents. Sankpal et al. ( 1999 ) reported acapillary-based vertical fabric support for A. niger that canrun for a period of 61 days with 120 140 g/l emerging GA.Later, Sankpal and Kulkarni ( 2002) found that the optimum biomass for efficient GA bioconversion on a porouscellulose support was 0.234 mg/cm 2 . Yeast-like strain A. pullulans was immobilized on porous-sintered glass andapplied in a fluidized bed reactor under defined fermenta-



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tion medium using 450 g/l glucose; this produced 315g/l GA in a continuous chemostat culture after 21 h(Anastassiadis and Rehm 2006b).

To develop a semicontinuous GA-production methodutilizing unconventional carbohydrate sources, Vassilev et al. (1993) studied whole-cell immobilization of A. niger onPUF in hydrol-based medium; the highest GA concentra-tion achieved was about 143 g/l. Whole-cell immobilizationof A. niger in calcium alginate using potassium ferrocya-nide-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 Panda1994). In a detailed study, Singh ( 2000) tested a variety of alternative carbohydrate sources such as sugarcane molas-ses, grape must, and banana must for semicontinuous production of GA using PUS- and calcium-alginate-immobilized whole cells of A. niger . Semicontinuous GA production with 98% yield was achieved using grape must with PUS-immobilized A. niger (Singh 2000). PUF-supported A. niger produced 92 g of GA from 1 ldeproteinized whey supplemented with lactose and glucosethan 69 g of GA from free mycelia (Mukhopadhyay et al.2005). Much work remains to be done concerning the useof PUS- and Ca-alginate-matrices with unconventionalcarbohydrate sources for continuous GA production.

Product recovery

Obtaining the desired product in pure form after fermenta-tion with alternative carbohydrate sources may be one of the tedious tasks for fermentation industries. The presenceof neutralizing agents such as calcium and/or sodium ionsis 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. FreeGA can be recovered from calcium gluconate either byclassical calcium sulphate precipitation or by passing thesolution through a column containing a strong acid-cationexchanger to absorb the calcium ions (Milson and Meers1985). Purified Glucono- -lactone crystals can be obtainedfrom supersaturated solutions of GA at 30 70C. Blom et al. (1952) recovered 45% solids of sodium gluconate bysimply concentrating the filtered fermentation broth, thenadding sodium hydroxide to pH 7.5 and drum drying. Avariant method of recovering GA from a clarified anddecolorized broth containing sodium gluconate has been patented whereby the solution is passed through a stronganion exchanger such as the Amberlite IRS400, whichretains the GA in the column.

The classical GA-recovery process was found particu-larly suitable for use with impure liquors derived from

rectified grape must (Singh 2000). Some loss of GA wasobserved during decolorization, yielding free GA with 89%of the total recovery. Thus, 120 g/l equivalent glucose fromrectified grape must results in 94 g of pure GA from thefermented broth after 10 days of SSF (Singh 2000).Therefore, the classical methods of separating purified GAwould be an advantage to the fermentation industries, withno extra expenditure in downstream processing.

Conclusion

GA production is a simple oxidation process that can becarried out by multiple modes of reaction; microbialfermentation has become a viable mode for commercial production. To make the traditional fermentation processmore economical, researchers have evaluated cheaper carbohydrate substrates and made innovative modificationsto the process. Among agro-food by-products, sugarcanemolasses and grape must can be used as alternativecarbohydrate substrates to produce GA under SSF on anindustrial scale, which will eventually pave the way to acheaper 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 byRashmi Singh for preparing this manuscript is gratefully acknowl-edged. We also thank the reviewers and the editorial team for their insightful suggestions regarding the review content.

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