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Biotechnology Advances 26 (2008) 293–303
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Biotechnology Advances
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Research review paper
Microbial production of dihydroxyacetone
Ruchi Mishra, Seema Rani Jain, Ashok Kumar ⁎Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, 208016-Kanpur, India
⁎ Corresponding author. Tel.: +91 512 2594051; fax: +E-mail address: [email protected] (A. Kumar).
0734-9750/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2008.02.001
A B S T R A C T DDD
A R T I C L E I N F OArticle history:
Dihydroxyacetone is extens Received 9 October 2007Received in revised form 3 January 2008Accepted 3 February 2008Available online 14 February 2008Keywords:Dihydroxyacetone productionGluconobacter oxydansBatch modeFed-batch modeSubstrate inhibitionProduct inhibition
ively used in cosmetic industry as an artificial suntan besides having clinical andbiological applications. Thus, it is important to meet the commercial demand of dihydroxyacetone at aneconomical and qualitative level. Microbial route of production is found to be more favorable fordihydroxyacetone as compared to chemical methods. This review gives detailed information about themicrobial route of dihydroxyacetone production. Till date the microorganism which is most utilized fordihydroxyacetone production is Gluconobacter oxydans. Some limitations associated with dihydroxyacetoneproduction by G. oxydans like substrate inhibition, product inhibition and oxygen limitation are discussedhere. Various fermentation modes and culture conditions have been tried for their ability to overcome theselimitations. It has been found that fed-batch mode of fermentation provides a better yield as compared tobatch mode for dihydroxyacetone production. Two-stage repeated fed-batch mode of fermentation has beenfound to be the most optimized mode. Immobilization has also been recognized as a much better alternativefor fermentation since it avoids the problem of substrate and product inhibition to a greater extent. Althoughthese methods have increased the dihydroxyacetone production to a prominent level yet the production hasnot reached the level required to meet the commercial demand. One looks for future prospects of developingrecombinant microbial method for dihydoxyacetone production.
© 2008 Elsevier Inc. All rights reserved.
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Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2932. Applications of dihydroxyacetone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2943. Chemical vs. microbial route for dihydroxyacetone production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2954. Microbial sources for dihydroxyacetone production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2965. Physiology of microbial synthesis of dihydroxyacetone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
5.1. Glycerol dehydrogenase enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2985.2. Dihydroxyacetone synthase enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
6. Culture conditions and fermentation modes utilized for dihydroxyacetone production . . . . . . . . . . . . . . . . . . . . . . . . . . . 2996.1. Batch mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2996.2. Fed-batch mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
7. Estimation of dihydroxyacetone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3008. Role of intracytoplasmic membrane in glycerol oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3019. Problems related to dihydroxyacetone production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
9.1. Inhibition on G. oxydans caused by glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3019.2. Inhibition on G. oxydans caused by dihydroxyacetone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
10. Conclusion and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302RETRA
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91 512 2594010.
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1. Introduction
Since the dawn of civilization, it has been human endeavor to makelife as comfortable as possible. Biotechnology is the outcome of man'svigorous efforts in the field of science in achieving this goal. Scientistsareworking hard to find applications of various research breakthroughs
Fig. 1. Chemical structure of dihydroxyacetone (DHA).
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in day-to-day life. A number of commercially important products havebeen identified so far, one of them is dihydroxyacetone (DHA) (C3H6O3).It is a simple, achiral and non-toxic sugar; formed under prebioticconditions by condensation of three molecules of formaldehyde (Erniet al., 2006). It is often derived from plant sources such as sugar beetsand sugar cane, by the fermentation of glycerin.
German scientists first recognized dihydroxyacetone (Fig. 1) as askin-coloring agent in the 1920s through its use in the X-ray process. Itwas noted that dihydroxyacetone caused the skin surface to turnbrown when spilled. Later as World War II began, further research inthis area temporarily halted, as scientists contributed their resourcesto the war effort. In the 1950s, Eva Wittgenstein at the University ofCincinnati did further research with dihydroxyacetone. Her studiesinvolved using dihydroxyacetone as an oral drug for treating childrenwith glycogen storage disease (Wittgenstein and Berry, 1960). Thechildren received large oral doses of dihydroxyacetone, and some-times spit or spilled the substance onto their skin. Healthcare workersnoticed that the skin turned brown after a few hours of dihydrox-yacetone exposure. Eva Wittgenstein continued to experiment withthis unique substance, painting dihydroxyacetone liquid solutionsonto her own skin. She was able to consistently reproduce thepigmentation effect, and noted that dihydroxyacetone did notpenetrate beyond the stratum corneum, or dead skin surface layer.
Coppertone introduced the first consumer sunless tanning lotioninto the marketplace in the 1960s. This product was called “quick tan”or “QT”. Since then, various methods are used for the production ofdihydroxyacetone in larger amount and at cheaper rates because of itscommercial value. In the 1970s, the food and drug administration
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Fig. 2. The reaction of dihydroxyacetone with the stratum corneum layer of the skinbased on Maillard reaction.
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added dihydroxyacetone to their list of approved cosmeticingredients.
2. Applications of dihydroxyacetone
Dihydroxyacetone is a very important chemical product. It is usedextensively in the cosmetic industry for making artificial suntans. Fairskinned people should limit their exposure to sun because of thehazardous consequences related to suntan, e.g., malignant melanoma,increase in wrinkles, immune suppression, cataract, etc. A naturalsuntan leads to skin cancer by the activation and deactivation ofvarious downstream signals in different signaling cascades, whichfinally lead to tumorigenesis. These findings indicate that a naturalsuntan is undesirable due to the risk of skin cancer associated with it.Therefore, it can be noticed that there is an increasing demand ofartificial suntan. These artificial suntans are based on dihydroxyace-tone, as an active ingredient (Brown, 2001). Dihydroxyacetone givesthe same tanning effect as the natural suntan but the difference lays inthe mechanism of action, due to which dihydroxyacetone gives atanned look without the risk of skin cancer. Depending on thedarkness of the tan required and the place of application, sunlesstanning products usually contain 2–5% dihydroxyacetone. The price ofsunless tanning lotions, creams, towellette, gels, mousse, foam, etcrange from $5 to $150/100 g, at present. It increases with the form ofthe sunless tanning substance, i.e., cream, gel, etc and also with theadditional functionalities e.g., walnut extract, SPF (Sun ProtectionFactor), antioxidants, vitamins, etc. Although these artificial suntanscause only temporary darkening of skin and need to be applied atregular intervals to maintain the tanned look. Dihydroxyacetonereacts with the amino acids present on the uppermost layer of skin,i.e., stratum corneum to produce a darkening effect. Themechanism ofdihydroxyacetone action is based on Maillard reaction, according towhich carbohydrates react with amino acids of proteins to produce agolden to brownpigmentation. The reaction of dihydroxyacetonewiththe stratum corneum layer is shown in Fig. 2. The intensity of colordeveloped is directly proportional to the concentration of dihydrox-yacetone used for pigmentation. Thus, dihydroxyacetone provides asunless tanning, i.e., tanning without the harmful effects of UV rays.Due to the penetration only up to stratum corneum i.e., dead layer ofskin, dihydroxyacetone does not cause any harmful effects on the skin.Earlier dansyl chloride was used as a fluorescent marker to assess thein vivo turnover of stratum corneum but various clinical andexperimental problems were found to be associated with it (Forestet al., 2003). Dihydroxyacetone has been proposed to be potentalternative to dansyl chloride becausemany of the problems related todansyl chloride are not found when dihydroxyacetone is used for thesame purpose.
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Fig. 3. A picture showing the high degree of sunburns caused on the skin of patientssuffering with vitiligo disease. (Courtesy — http://dermnetnz.org/treatments/dihydrox-yacetone.html).
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Due to potential capacity of dihydroxyacetone to cause pigmenta-tion, it is also used for the treatment of vitiligo (Fig. 3), an autoimmunedisease in which pigment cells (melanocytes) are destroyed; resultingin irregularly shapedwhite patches on the skin. This method of vitiligotreatment is practical and well accepted (Fesq et al., 2001). It is alsoused for protection against disease called variegate porphyria(Asawanonda et al., 1999; Levy, 2001), such patients can be benefitedby the use of dihydroxyacetone based products as their skin is overlysensitive to sunlight and dihydroxyacetone can provide someprotection against UVA. This is due to the melanoidin pigmentproduced by their reaction with skin, which resembles melanin(Nguyen and Kochevar, 2003; Ahmed, 2002; Fusaro and Rice, 2005).Although dihydroxyacetone has not been accepted as a sunscreen, yeta study (Petersen et al., 2003) reveals that topical application ofdihydroxyacetone in high concentrations on the hairless miceirradiated with moderate UV doses may lead to a delay in skin cancerdevelopment. Dihydroxyacetone has been found to show betterresults for sun protection when used along with napthoquinone.Another potential use is for enhancement of photo chemotherapywith PUVA (Psoralen+UVA) treatment for stable plaque psoriasis.
Dihydroxyacetone is used in the chemical industry as a versatilebuilding block (Hekmat et al., 2003) for the synthesis of a variety offine chemicals. For example, dihydroxyacetone has been used for theproduction of lactic acid (Bicker et al., 2005) and 1, 2 propylene glycol.Due to the non-toxic nature of dihydroxyacetone, it is also used for thesynthesis of biomaterials with non-toxic degradation products(diblock polymers) for many applications including drug delivery.Dihydroxyacetone is also used for preparation of natural rearrange-ment products, which can play significant biological roles. It has beenfound that dihydroxyacetone and glycine Maillard reaction is used forthe formation of Heyns rearrangement products (HRPs) (Shipar,2006). Dihydroxyacetone can affect the quality of wine by affectingthe mouth feel of the wine, leading to a sweet/etherish property. Thishappens due to conversion of glycerol (which enhances the mouthfeel of wine) to dihydroxyacetone by the acetic acid bacteria present inwine. Dihydroxyacetone can also affect the anti-microbial activity inwine due to its ability to bind SO2 (Toit and Pretorius, 2002). Onreactionwith proline dihydroxyacetone produces a “crust-like” aroma(Drysdale and Fleet, 1988). Dihydroxyacetone has also been used tofind the impaired metabolic step, which leads to diabetic beta cells.The researchers investigated insulin secretory capacity by stimulationwith dihydroxyacetone (Tsuura et al., 1994). Dihydroxyacetone isextensively used in medical industry as well. It has been found to havean antidotal effect towards cyanide poisoning. It was found that oraldihydroxyacetone dose of 2 and 4 g/kg, given to mice 10 min beforeinjection (i.p.) of cyanide increased the LD50 values of cyanide from5.7 mg/kg to 12 and 17.6 mg/kg, respectively (Niknahad andGhelichkhani, 2002). The antagonizing effect of dihydroxyacetonewas increased particularly when administered in combination withanother cyanide antidote, i.e., sodium thiosulphate (Niknahad andO'brien, 1996), also the convulsions that occurred after cyanide
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Table 1Comparison of the microbial and chemical route used for dihydroxyacetone production
S. No. Property Microbial route
1. Cost Economical2. Environmental concern It is an ecofriendly process.3. Isolation and purification
of dihydroxyacetoneIt is easier to isolate and purify dihydroxby this route without much significant e
4. Catalyst The catalyst employed in this process iscatalyst (i.e., enzymes) hence they are n
5. Overoxidation Enzymes are very specific for their substspecific products hence chances of overo
6. Commercial status This process is used for commercial prodof dihydroxyacetone.
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intoxication were prevented by its use. Dihydroxyacetone has alsobeen proposed to be involved in weight augmentation and fat loss,antioxidant activity, increasing endurance capacity (Stanko et al.,1990; Ivy, 1998; Schlifke, 1999). A bi-functional channel, i.e.,aquaglyceroporin, of Plasmodium falciparum (PfAQP) facilitates in thepermeation of glycerol for glycerolipid biosynthesis and also functionssupposedly in the osmotic protection of parasite. This channel haspermeability for water and solutes. The activity of P. falciparumglyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) was checkedfor inhibition by dihydroxyacetone. It was found that 2.5 mMdihydroxyacetone abolished PfGAPDH activity within 6 h (Djuranovicet al., 2006). Thus, the permeability of aquaglyceroporin for glycolyticmetabolites, like, dihydroxyacetone may be of physiological signifi-cance for synthesis of antimalarial drugs. Similarly, dihydroxyacetonealso has antiproliferative effect on Trypanosoma brucei bloodstreamforms. It causes up to 70% cell cycle arrest in the G2/M phase at aconcentration of 2 mM, also dihydroxyacetone treated parasites showsymptoms such as ultrastructural alterations, including an increase ofvesicular structures within the cytosol and the presence of multi-vesicular bodies, myelin-like structures, autophagy-like vacuoles anddisorder of the characteristic mitochondrion structure. Based on suchinformation, dihydroxyacetone has been considered as a significantmolecule for designing of new trypanosomal drugs (Uzcategui et al.,2007). Also certain antiviral novel fluorocyclopropyl nucleosides havebeen synthesized using dihydroxyacetone as a starting point, out ofthese some of the nucleotides have shown significant antiviral activity(Oh and Hong, 2007).
3. Chemical vs. microbial route for dihydroxyacetone production
After 1950, as the petrochemical industry emerged as adeveloping industry, microorganisms lost their significance as ameans for synthesis of chemicals (Nagasawa and Yamada, 1995).This was due to the fact that microorganisms could not producechemicals at a commercially acceptable scale. Nowadays, thisscenario has changed and microbial processes are emerging asstrong competitors of chemical processes for the synthesis ofproducts for the chemical or pharmaceutical industries. Chemicalindustries are currently facing the demands of energy efficiency,conservation of natural resources and reduction of environmentalpollution. These demands can be better fulfilled by the introductionof microbial route into production. Certain commercially availablesynthetic products are obtained from microbial sources by the use ofvarious biotechnological approaches. Chemical route cannot pro-duce certain products economically such as, fine chemicals. Some-times, extensive safety measures are required due to the hazardouschemical reactions involved in a chemical process. To avoid thismicrobial processes are preferred due to their specificity and mildprocess environment.
The literature study reveals that dihydroxyacetone can bechemically produced via two different sources of substrate;
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Chemical route
CostlyIt may be hazardous.
yacetonefforts.
Isolation and purification of dihydroxyacetoneis difficult in the case of chemical route.
biologicalon-poisonous.
They can be poisonous.
rate and producexidation are rare.
Overoxidation is a matter of concern in chemical process.
uction Although at laboratory scale experiments are done via thisroute but it has yet not reached the commercial level.
Table 2Glycerol-oxidizing activity of intact cells of Acetobacter and Gluconobacter
Microorganism Total activity (μmol/min/ml of broth) Specific activity (μmol/min/mg of cells)
Acetobacter suboxydans ATCC 621 6.20 1.02Acetobacter xylinum A-9 7.95 1.07Gluconobacter melanogenus IFO 3293 4.99 0.66Gluconobacter melanogenus IFO 3294 4.21 0.64
Table 3List of microorganisms producing dihydroxyacetone
S. No. Name of the strain
1. Sorbose bacillus/Bacillus xylinum2. Acinetobacter sp. strain JC1 DSM 38033. Acetobacter xylinum A-94. Gluconobacter melanogenus IFO 32935. G. melanogenus IFO 32946. Hansenula polymorpha CBS 47327. Candida boidinii 22018. Klebsiella aerogenes9. Escherichia coli (strain ECFS)10. Gluconobacter oxydans ATCC 62111. Pichia membranifaciens12. Bacillus licheniformis B-0557113. Acetomonas sp.
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formaldehyde and glycerol. Chemical production of dihydroxyacetonefrom formaldehyde is based on a condensation reaction called as“formoin reaction”. Josep Castells and co-workers first described thisreaction in 1980, according to which condensation of formaldehyde inthe presence of conjugate bases of thiazolium ions (thiazolium ylides)yields a complex mixture of branched and unbranched aldehydes andketones (Castells et al., 1980). A process based on this methodobtained dihydroxyacetone yield of 82%. The problem associated withthis method is difficulty in purifying dihydroxyacetone, which alsoadds to the cost.
The production of dihydroxyacetone from glycerol via chemicalmeans can be done either catalytically or electrocatalytically. Glycerolis a highly functionalized molecule, which can yield different kinds ofproducts in the presence of specific catalytic conditions. It has beenfound that by controlling the parameters like temperature, pH, and bythe use of precious metals like Pt, Pd, etc as catalyst, the orientation ofglycerol oxidation can be directed towards either primary alcoholfunctions or secondary alcohol functions. Orientation towardssecondary alcohol functions, i.e., towards dihydroxyacetone produc-tion, can be directed in an efficient way by the use of Bi doped Ptcatalyst (Dimitratos et al., 2005; Bianchi et al., 2005). Around 50–70%conversion was obtained by this method (Garcia et al., 1995). The useof Pt metal has a drawback, since it causes poisoning. On the otherhand, electrocatalytic oxidation of glycerol can be done by simplyapplying an electric potential (1.1 V vs. Ag/AgCl) to a glycerol solutionin the presence of 15-mol% TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) maintained at a pH of 9.1. The oxidation via this method can bedone in a single pot itself and also this method is waste free. It wasfound that after 20 h of reaction, 25% yield of dihydroxyacetone wasobtained during electrocatalytic oxidation of glycerol (Ciriminna et al.,2006). Upon extension of the reaction time, overoxidation of thedihydroxyacetone occurs and hydroxypyruvic acid is formed. Thus,this process is also not very advantageous.
Commercial synthesis of dihydroxyacetone is done more econom-ically via microbial route due to the expensive safety measurementsrequired in case of chemical route (Hekmat et al., 2003). Table 1compares the two routes of microbial production and explains howthe microbial route of dihydroxyacetone production is more efficientas compared to that of chemical route. It has recently been reportedthat the enzyme catalyzed chemical bioconversions are moreacceptable by the pharmaceutical and chemical industries as apractical alternative to chemical synthesis methods due to theintractable synthetic problems involved with the chemical synthesismethods and also because of stringent environmental constraints.This particularly applies to the production of emerging classes oforganic compounds that are the targets of molecular and biomedicalresearch (Koeller and Wong, 2001).
4. Microbial sources for dihydroxyacetone production
Bertrand first observed production of dihydroxyacetone fromglycerol through bacterial route in the year 1898. He used bacteriacalled Sorbose bacillus, the bacteria was able to oxidize certainsecondary alcohols to their corresponding ketone sugars. Later thisS. bacillus was found to be identical with the acetic acid bacteriumBacillus xylinum. Dihydroxyacetone was produced due to the activity
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of the enzyme dihydroxyacetone synthase (DHAS) found in acarboxydobacterium, Acinetobacter sp. strains JC1 DSM 3803, whenit was grown on methanol (Young et al., 1997). The enzyme produceddihydroxyacetone from formaldehyde and xylulose 5-phosphate assubstrate.
Intact cells of the following microorganisms; Acetobacter subox-ydans ATCC 621, Acetobacter xylinum A-9, Gluconobacter melanogenusIFO 3293, and G. melanogenus IFO 3294 were studied for their glyceroloxidizing activity (Nabe et al., 1979). A. xylinum A-9 showed thehighest activity out of them (Table 2). Thus, this shows that A. xylinumhas the highest capability to produce dihydroxyacetone out of thesebacteria. Mutant of methanol-utilizing yeast, Hansenula polymorphaCBS 4732 can produce a significant amount of dihydroxyacetone usingmethanol as a substrate (Kato et al., 1986), in a resting-cell reaction.This mutant is impaired in its ability to synthesize the enzymedihydroxyacetone kinase.
Due to the ketogenic action of Acetomonas strains, they canproduce dihydroxyacetone from glycerol, 49 out of 50 strainsproduced dihydroxyacetone abundantly (Carr and Shimwell, 1961).Similarly, dihydroxyacetone can also be produced from yeast Candidaboidinii 2201 through the enzymatic action of dihydroxyacetonesynthase (Streekstra et al., 1987), a key enzyme for carbon compoundassimilation in this yeast. This enzyme uses formaldehyde as the mainsubstrate while methanol was used for concentration of dihydrox-yacetone. Klebsiella aerogenes follows two pathways for glyceroldissimilation based on the presence or absence of oxygen (Neijsselet al., 1975). During anaerobic conditions the culture invariablycontained glycerol dehydrogenase enzyme. Although this microor-ganism is not used as a direct source of dihydroxyacetone productionrather it leads to DHAP (dihydroxyacetone phosphate) production.
Escherichia coli (strain ECFS) was also found to produce a DPNlinked glycerol dehydrogenase (Asnis and Brodie, 1953). This enzymewas sufficiently heat stable to be isolated in a highly active state whenthe cell free extract was subjected to a heat treatment to remove theother oxidative enzymes produced. Evidently, glycerol oxidation in thepresence of this enzyme produced dihydroxyacetone in a 1:1 molarratio with the DPN+ reduced in a specifically DPN linked reaction.Gluconobacter oxydans, formerly called as A. suboxydans (Batzing andClaus, 1971), is the most extensively used microorganism in the
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present day research for dihydroxyacetone production. It is a gram-negative bacterium; obligate aerobe belonging to family Acetobacter-aceae (Gupta et al., 2001) (found only in flowers and fruits). Itproduces dihydroxyacetone via incomplete oxidation of glycerol withthe activity of enzyme glycerol dehydrogenase (Deppenmeier et al.,2002). Recently, a microorganism from soil sample named as, Pichiamembranifaciens, has also been used for dihydroxyacetone production.It was found that at temperature 29.6 °C and pH 6.74, the maximumconcentration of dihydroxyacetone obtained was 12.91 g/l when theduration of growth was 48.8 h (Liu et al., 2007). Various microorgan-isms used for dihydroxyacetone production are listed in Table 3.
5. Physiology of microbial synthesis of dihydroxyacetone
G. oxydans is a gram-negative, rod or oval shaped bacteriawith sizefrom about 0.5 to 0.8 mm×0.9 to 4.2 mm. It has the ability to partiallyoxidize carbon substrates like carbohydrates and alcohols e.g, D-sorbitol, glycerol, D-fructose, and D-glucose, through the process ofoxidative fermentation, for synthesis of compounds like Vitamin C, D-gluconic acid, dihydroxyacetone, ketogluconic acid, xylitol, vinegar,etc (Macauley et al., 2001). G. oxydans bacteria are naturally found inflowers, fruits, garden soil, alcoholic beverages, cider, and soft drinks.These strains grow at such places because they are capable of growingin high concentrations of sugar solutions and low pH values (optimalpH for growth is 5.5–6.0) (Gupta et al., 2001). The genome size ofGluconobacter is small ranging about 2240 to 3787 kb, totalnumber of genes and DNA molecules is 2664 and 6 respectively(Prust et al., 2005). The total size of all the DNA moleculesis 2,922,384 bp and its G+C content is 61%. The optimum temperaturefor its growth is 25 to 30 °C and it cannot withstand high temperaturesbeyond 37 °C. G. oxydans being an obligate aerobe can derive itsenergy via broadly two methods, i.e., either by oxidation of sugars,aliphatic and cyclic alcohols, and steroids to oxidation products orthrough the pentose phosphate pathway where phosphorylationoccurs initially then proceeds with oxidation through the pathway.
Fig. 4. A graphical image describing the physiology of the bi
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Since the carbon dioxide produced from glucosewas from the pentosephosphate pathway, this was suggested that G. oxydans has anincomplete set of tricarboxylic acid cycle (TCA) enzymes (Gätgenset al., 2007). These bacteria carry out incomplete oxidation of variouscarbon substrates by the help of membrane bound dehydrogenases.These strains are non-pathogenic to humans or animals, but theycause bacterial rot to apples and pears turning them to shades ofbrown (Gupta et al., 2001).
Glycerol dehydrogenase enzymes are of two types, one ismembrane bound which contains a novel prosthetic group, pyrrolo-quinoline quinone (PQQ), as in the cases of alcohol, aldehyde, and D-glucose dehydrogenases of acetic acid bacteria; another one ispyridine nucleotide dependent, which has been partially purifiedfrom the soluble fractions of E. coli (Asnis and Brodie, 1953), A.suboxydans and Klebsiella pneumonia. Isolation, purification andproperties of these enzymes have been studied in detail by variousresearchers. The structure, specificity, and mechanism of an NADdependent glycerol dehydrogenase, i.e., Family III polyol dehydrogen-ase of Bacillus stearothermophilus have been reported (Ruzheinikovet al., 2001). On the other hand, solubilization, purification andproperties of membrane bound glycerol dehydrogenase have beendescribed in a strain of Gluconobacter, i.e., Gluconobacter industriusIFO 3260. The purification of this enzyme was done throughfractionationwith polyethylene glycol 6000. The activity of membranebound glycerol dehydrogenase enzyme was completely bleachedwhen treated with 2 mM EDTA (pH 7.5). This activity was restored tothe original level when cofactor (prosthetic group) of membranebound glycerol dehydrogenase enzyme, i.e., pyrroloquinoline quinone(PQQ) was added in the presence of magnesium ions. Methanol wasused to isolate these prosthetic groups. Thus, the membrane boundglycerol dehydrogenase enzyme was confirmed to be a quinoproteinin which pyrroloquinoline quinone functioned as the prostheticgroup (Ameyama et al., 1985). Similarly, the properties andanalytical application of PQQ-dependent glycerol dehydrogenasefrom Gluconobacter sp. 33 was also studied in detail. The purified
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ochemical activities occurring in Gluconobacter oxydans.
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glycerol dehydrogenase was found to show an optimum activity at pH7.0–7.5 and was most stable at pH 8.5–9.5; also the rate of glyceroloxidation was maximum at 33–37 °C. The Km values for differentsubstrates, i.e., glycerol, D-sorbitol, and D-mannitol were 0.83, 1.0, and2.4 mM, respectively (Lapenaite et al., 2005). Such PQQ-dependentmembrane bound glycerol dehydrogenase can also be used forbiosensor application for detection of glycerol and triglycerides inreal wine samples (Ameyama et al., 1985; Lapenaite et al., 2005). Theactivity of these periplasmically localised PQQ-dependent membranebound dehydrogenases has been found to increase by an application ofa flexible polyvinylimidazole osmium functionalized polymer forefficient electrical wiring of whole G. oxydans cells. The sensitivity ofthe enzyme by such application was found to be similar to that ofsensitivity profile for ferricyanide as soluble mediator (Vostiar et al.,2004).
Two of the enzymes linked with dihydroxyacetone production areglycerol dehydrogenase and dihydroxyacetone synthase. The physiol-ogy of dihydroxyacetone production varies on the basis of thefollowing parameters:
Substrate source: Glycerol (Kato et al., 1986; Jones and Bellion,1991), methanol (Carr and Shimwell, 1961) and formaldehyde(Streekstra et al., 1987).Microorganism source: Various microorganisms are used fordihydroxyacetone production (described in Section 4).Enzyme physiology: Enzyme may be heat labile or heat stable (e.g.,in E. coli).
5.1. Glycerol dehydrogenase enzyme
Dihydroxyacetone synthesis by the activity of glycerol dehydro-genase enzyme is well illustrated by the metabolism of glycerol inbacteria G. oxydans. The graphical representation of biochemicalactivities happening in G. oxydans during glycerol dissimilation wasdescribed earlier as shown in Fig. 4 (Ohrem and Voβ, 1996a,b). Exactphysiology of dihydroxyacetone formation during glycerol metabo-lismwas explained by Claret et al. (1992). This microorganism utilizes
Fig. 5. Different metabolic pathways used for gly
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two catabolic pathways for glycerol dissimilation. Here, glyceroldehydrogenase is membrane bound and it is the only enzymeresponsible for dihydroxyacetone production. It produces dihydrox-yacetone without NADHmediation employing O2 as the final acceptorof reduced equivalents (Claret et al., 1994). Since the enzyme ismembrane bound, dihydroxyacetone is directly released into theculture. G. oxydans lacks glycolytic and carboxylic acid pathways,hence the pathway involving membrane bound glycerol dehydrogen-ase is indispensable for its energy requirements. On the other handcytoplasmic pathway begins with phosphorylation of glycerol fol-lowed by dehydrogenation to DHAP. This pathway allows for bacterialgrowth. DHAP produced is catabolized by pentose phosphate path-way. The pathway for dihydroxyacetone production is shown in Fig. 5.The genomics information about glycerol dehydrogenase fromG. oxydans is discussed as follows (information obtained from UniProtKnowledgebase):
Glycerol dehydrogenase large subunit precursor (EC 1.1.99.22)(Gluconate/polyol dehydrogenase large subunit) (D-sorbitol dehydro-genase subunit sldA) (SLDH) (D-arabitol dehydrogenase large subunit)(ARDH)
Gene: sldA (ga5dhB) (GOX0854)Length of protein chain: 743Organism: G. oxydans (Gluconobacter suboxydans)Glycerol dehydrogenase small subunit (EC 1.1.99.22) (Gluconate/
polyol dehydrogenase small subunit) (D-sorbitol dehydrogenasesubunit sldB) (SLDH) (D-arabitol dehydrogenase small subunit)(ARDH)
Gene: sldB (g5dhA) (GOX0855)Organism: G. oxydans (Gluconobacter suboxydans)Length of protein chain: 126
5.2. Dihydroxyacetone synthase enzyme
Dihydroxyacetone production via this enzyme can be discussedthrough the illustration of dihydroxyacetone production in themutant(No. 65) of yeastH. polymorpha CBS 4732 (Jones and Bellion,1991). Theproduction of dihydroxyacetone from methanol in H. polymorpha
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cerol assimilation in Gluconobacter oxydans.
Table 4Characteristic parameters of batch cultivations of Gluconobacter oxydans in differentmedia
Property Complex media Semisynthetic media
Initial glycerol (g/l) 51 31 55 29.3y (dbm/glyc) (g/g) 0.039 0.052 0.016 0.02µmax (l/h) 0.3 0.38 0.24 0.38qDHA,max (g/(g.h)) 6.3 7.8 10.3 10.4
Yield of dry biomass on glycerol, y (dbm/glyc); specific growth rate, μmax and maximalspecific dihydroxyacetone production rate, qDHA,max.
Table 6Fermentation parameters obtained during fed-batch mode of fermentation
Parameter of fermentation Fed-batch culture
ydha/glycerol 0.85 mol DHA/mol glycerolDHA 108.5 gl−1
Culture length 40 hrx max 0.3 g l−1 h−1
rs gmax 7.7 g l−1 h−1
rs mmax –
rp dmax 7.2 g l−1 h−1
rp fmax –
µmax 0.33 h−1
qs gmax 12 h−1
qs mmax –
qp dmax 10 h−1
qp fmax –
Experimental conditions: pH=6: temperature=28 °C; agitation=800 rpm; aeration=1 vvmlml. Inoculation, 5.9% v/v by 1-day-old culture; agitation, 800 rpm; aeration, 1 vvm; pH, 6;temperature, 28 °C.
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occurs in peroxisomes. Mutant no. 65 of this yeast lacks dihydrox-yacetone kinase enzyme required for the conversion of dihydrox-yacetone to DHAP (dihydroxyacetone phosphate). This leads to thesignificant accumulation of dihydroxyacetone through DHAS (dihy-droxyacetone synthase) activity. Although, an initial step of enzymeinduction is necessary for effective production of dihydroxyacetonefrom methanol (Carr and Shimwell, 1961). Cells of no. 65 mutantwithout the enzyme induction step produced only a small amount ofdihydroxyacetone.
6. Culture conditions and fermentation modes utilized fordihydroxyacetone production
Various factors were found to be involved in enhancing the growthconditions during G. oxydans culture. The media generally used fordihydroxyacetone production include glycerol, mineral salts andcertain amino acids as its constituent. The growth factors requirementfor this bacteriumwas studied and it was found that pantothenic acidwas essential for growth and p-aminobenzoic acid was also required.Sufficient organic nitrogen requirement for nutrition of the bacteriawas fulfilled by hydrolyzed casein. This was evidenced by the fact thatwhen mixture of amino acids was replaced by hydrolyzed casein, itshowed excellent growth of bacteria (Underkofler et al., 1943).
A semisynthetic culture medium was designed for growth anddihydroxyacetone production by G. oxydans. Incorporation of justthree essential components of vitamins (pantothenate, p-aminobenzoic acid and nicotinic acid) and two amino acids (serine,glutamine) reduced the yeast extract concentration by 5–10% of theprevious concentration (Wethmar and Deckwer, 1999). This newsemisynthetic media gave a lower yield and comparable growth but ahigher productivity as compared to that of media generally used fordihydroxyacetone production (Table 4).
6.1. Batch mode
Bertrand first isolated dihydroxyacetone from the metabolismsolution in 1904. Later A. suboxydans (G. oxydans) was used fordihydroxyacetone production using a culture medium containing 4.0–5.5% solution of glycerol in a 3% yeast extract. The pH was adjusted to5.2 and the culture was carried out for around 14 days during whichfermentation came to an end. The yields obtained are given in Table 5.Similarly, for initial glycerol concentrations of 50 and 100 g/l, BoriesRETR
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Table 5Yield of dihydroxyacetone from glycerol during batch culture
S. No. pH 0.153 cc KMnO4/2.0 cc metabolism solution
1 2.80 12.22 Not determined 12.53 2.88 13.04 Not determined 12.35 Not determined 12.2
et al. (1991) reported that the dihydroxyacetone yield in batchcultivation was 87 and 90%, respectively (Virtanen and Nordlund,1933).
6.2. Fed-batch mode
In this operational mode of fermentation a high concentration ofdihydroxyacetone can be obtained while maintaining a near constantand optimal (non-limiting and non-inhibitory) glycerol content: 25–30 g/l. The total production time for 108 g/l of dihydroxyacetone is40 h, while in the case of batch fermentations it is estimated to be60 h, which was necessary for the formation of 92.5 g/l ofdihydroxyacetone (Bories et al., 1991). The specific rates and maximalproductivity values also increased in the fed-batch mode (Table 6).
Later it was found that repeated fed-batch fermentation processfor the synthesis of dihydroxyacetone from glycerol using G. oxydanswas more useful as compared to conventional fed-batch cultures. Thisrepeated fed-batch process was optimized in the study done byHekmat et al. (2003). Cleaning, sterilization, and inoculation proce-dures could be improved significantly by this method as compared tothe conventional fed-batch process. A stringent requirement was thatthe product concentration was kept below a critical threshold level atall times in order to avoid irreversible product inhibition of the cells. Athreshold value of about 60 kg/m3 of dihydroxyacetone was obtained.To avoid the influence of product inhibition caused by dihydroxyace-tone during glycerol fermentation by G. oxydans a novel semicontin-uous two-stage repeated fed-batch process was examined (Baueret al., 2005). It was shown that the culture was able to grow up to adihydroxyacetone concentration of 80 kg/m3 without any influence ofproduct inhibition.
The focus of fermentation industry for dihydroxyacetone produc-tion is mainly on two aspects, one is to increase the biomassconcentration and the other one is to avoid the inhibition caused bythe product and substrate on the G. oxydans bacilli. At the same time itis highly desirable to achieve such properties at a minimum possiblecost. The amount of biomass directly corresponds to the amount of
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Dihydroxyacetone g in 1000 cc. Dihydroxyacetone % from glycerol
45.35 85.646.65 88.048.75 92.045.85 86.645.35 85.6
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available enzyme, i.e., glycerol dehydrogenase, for the conversion ofglycerol to dihydroxyacetone; therefore an increase in biomassconcentration is advantageous for dihydroxyacetone fermentation.The maximum cell density obtained was about 6×109 cells/ml (Whiteand Claus, 1982). G. oxydans is the strain of choice out of the 10 Glu-conobacter strains because it gives the best biomass yield in shakesflask culture. In a very recent study various parameters affectingbiomass production have been studied and a significant increase inbiomass production has been obtained. This study was carried out in1000 ml shake flasks with a volume of 200 ml at 26 °C and 150 rpmshaker speed. Carbohydrate sources like glucose, maltose, mannitoland sorbitol were checked for their ability to increase biomassproduction at a concentration range of 25–200 g/l and it was foundthat sorbitol was the best carbon source for achieving highest growth.A cell density of 4.3×1010 cells/ml was obtained during fermentationtime of 43 h, which was around 7 times more when compared to thecell density reported earlier. Various parameters like media composi-tion, pH, and bioreactor conditions were optimized during this studyto achieve such yield (Albin et al., 2007). Although the yield obtainedby above method is very high yet it will be desirable to obtain such ahigh yield at an economical level. A study reveals a method forproduction of dihydroxyacetone at a low cost from agriculturalbyproducts. Here, instead of sorbitol and yeast extract as mediumfor G. oxydans biomass production, corn meal hydrolysate and cornsteep liquor were employed. It was found that the yield ofdihydroxyacetone was almost comparable to that of a nutrient richmedium, but the cost of production was only 15% of that cultured insorbitol and yeast extract medium (Wei et al., 2007b). Thus, such aneconomical process can be of great use for commercial production ofdihydroxyacetone.
It is also necessary to understand the kinetics of glycerol oxidationfor a better control on the bioprocess conditions and fermentationperformance of dihydroxyacetone production. A lot of studies havediscussed the mathematical models explaining the kinetics of glycerolfermentation. In one such study influence of pH on the metabolism ofG. oxydans was explained via mathematical approach and it wasshown that product formation follows Luedeking–Piret kinetics(Ohrem and Voβ, 1995). Similarly some other studies for designingof fermentation process for dihydroxyacetone were conducted inwhich a structured segregated mathematical model was developedbased on kinetic measurements (Bauer and Hekmat, 2006). Thesemodels provided the information about the dynamics of batch andfed-batch fermentations with good agreement with measured con-centration profiles (Ohrem and Voβ, 1996a).
To avoid inhibition caused by the product and the substrate duringG. oxydans culture, the best way is to immobilize the cells onto asuitable polymer surface so that the cells do not come in direct contactwith the substrate and product. Cell immobilization can be defined asrestricting the cell mobility in a fixed space. Immobilization may leadto some important advantages such as cell reutilization and elimina-tion of product recovery and purification processes if the product isextracellular. It may also provide a better environment for cell activity.Cell immobilization can be done broadly in twoways, entrapment andsurface immobilization. The immobilization of cells can also havesome environmental applications. Such immobilized microbial cellscan be used extensively in various scientific studies and industrialapplications (Cassidy et al., 1996).
It was observed during a study that calcium alginate immobilizedG. oxydans cells maintained consistent oxidative activity withtemperature and pH during oxidation of glycerol to dihydroxyacetonewhereas in the case of free cells the activity was decreased(Adlercreutz et al., 1985). Similarly, a method of immobilization of G.oxydans cells in polyvinyl alcohol (PVA) has been developed veryrecently (Wei et al., 2007a). The use of immobilized cells forconversion of glycerol to dihydroxyacetonewas carried out repeatedlyin a 1.5 l stirred tank reactor and the average conversion rate obtained
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was 86%. The shape of PVA beads was not affected even after fiveconsecutive cycles and 90% initial activity of the biocatalyst wasrestored even after repeated use. The kinetics during immobilizationis different as compared to that of free cells, e.g., kinetics of glucoseoxidation by G. oxydans cells immobilized in calcium alginate wasdescribed in the 1980s (Tramper et al., 1983). Raska et al. have recentlydescribed kinetics of immobilized G. oxydans cells during dihydrox-yacetone production from glycerol. In this study biotransformation ofglycerol was carried out in an isothermal isochoric batch reactor, theG. oxydans were immobilized in poly vinyl alcohol gel capsules. Therate constants determined by this method can be advantageousduring designing of bioprocess model for industrial production (Raskaet al., 2007).
Gluconobacter is an obligate aerobe; hence, it requires very soundprovision for oxygen supply during fermentation so that a higher yieldof desired product can be obtained. It was found in G. melanogenus3293 that the conversion efficiency was increased over threefold if thepartial pressure of oxygen in the aerationwas increased in a controlledmanner (Flickinger and Perlman, 1977). Due to oxygen enrichment,the specific conversion of glycerol to dihydroxyacetone was increasedfrom 12.2 g of dihydroxyacetone/g of cell mass to a point of maximumconversion to 35.8 g.
A major drawback of immobilized whole cells in aerobic processesis the oxygen diffusion limitation in the system. Ca-alginateimmobilized cells were used as a model system for conversion ofglycerol to dihydroxyacetone. It had been reported that continuousproduction using a packed bed was increased almost twenty-fold inthe reactor when hydrogen peroxide was used as a source of oxygen(Hoist et al., 1982). Later it was checked if hydrogen peroxide couldimprove oxygen supply in the whole cell model system using G.oxydans ATCC 621 immobilized in ca-alginate. Organisms belonging tothe genus Gluconobacter have high catalase activity, thus catalyticdecomposition of hydrogen peroxide takes place inside the beadsthereby reducing the transport problems connected with oxygentransfer.
In another study for oxidation of glycerol in immobilized G.oxydans cells, oxygen supply was provided by p-benzoquinone as asubstitute for oxygen. It was found that in the presence of p-benzoquinone reaction rate was much higher as compared to whenoxygen was used alone (Adlercreutz and Mattiasson, 1984). Theeffectiveness of p-benzoquinone as compared to that of oxygen wasdue to the fact that it gives a higher maximal reaction rate and it ismore soluble in water than oxygen.
7. Estimation of dihydroxyacetone
Campbell has reported quantitative estimation of dihydroxyace-tone in the year 1926 (Campbell, 1926). Another method fordihydroxyacetone estimationwas based on the reaction of the ketonicgroup with resorcinol in concentrated hydrochloric acid to givecolored products. This method was based on Seliwanoff test forketoses. The intensity of color developed was proportional to ketoseconcentration (Tsao and Schwartz, 1962). Other researchers have donefurther modifications in this method. Aldoses and polyols are notfound to interfere with the estimation procedure but other ketosesmay interfere. Therefore, dihydroxyacetone can be well estimated bythis method even in the presence of glycerol and glyceraldehyde. Amodified enzymatic assay (based on glycerol assay) has also been usedfor the estimation of dihydroxyacetone and glyceraldehydes (Pinteret al., 1967). It follows the quantitative reduction to glycerol in thepresence of sodium borohydride. Dihydroxyacetone oxidation ofglycerol by bacterial cells of G. oxydans can also be monitored by abi-enzymatic biosensor that has high sensitivity. The enzyme used inthis method was galactose oxidase (Tkáč et al., 2001).
Although more appropriate method for dihydroxyacetoneanalysis in fermentation broth is via pyrolytic methylation-gas
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Table 7Comparison of characteristic parameters obtained during Gluconobacter oxydanscultures on glycerol at different concentrations
Glycerol (gl−1) 31 51 76 95 129Fermentation time (h) 12 20 30 48 78Producted biomass (gl−1) 1.62 1.98 1.95 1.90 1.85yx/s (%) 5.2 3.9 2.6 2.0 1.4Producted dihydroxyacetone (gl−1) 28.5 47 66 86 106yp/s (%) 92 92 87 90 82rx max (g l−1 h−1) 0.25 0.22 0.15 0.10 0.07rs max (g l−1 h−1) 5.76 5.14 4.56 3.79 3.54rp max (g l−1 h−1) 5.19 4.96 4.15 3.67 3.46µmax (h−1) 0.38 0.30 0.24 0.21 0.13qs max (g g−1 h−1) 8.73 7.49 6.75 3.07 2.76qp max(g g−1 h−1) 7.79 6.32 5.04 3.01 2.17
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chromatography (GC) using a vertical microfurnace pyrolyzer. Dihy-droxyacetone and glycerol were converted to their correspondingmethyl ethers by online pyrolytic methylation in the presence of anorganic alkali, i.e., tetramethylammonium hydroxide ((CH3)4NOH,TMAH). This method for dihydroxyacetone estimationwas found to berapid, convenient and highly sensitive (Lili et al., 2006). It could also beused for monitoring and control of bioprocesses of dihydroxyacetoneproduction and further studies will help this process to develop into amethod for dihydroxyacetone determination in the food and cosmeticsamples.
Dihydroxyacetone can also be analyzed by more subtle techniquessuch as HPLC (High Performance Liquid Chromatography). Dihydrox-yacetone undergoes a derivation reaction during analysis via HPLCmethod, this increases the efficiency of chromatographic process byenhancing the response and the detection limit (Ferioli et al., 1995).Another rapid method for the detection of dihydroxyacetone based onreversed phase HPLC with UV detection has been identified. In thismethod dihydroxyacetone is derivatized with penta-fluorobenzylhy-droxylamine (PFBHA) solution (Biondi et al., 2007). Dihydroxyacetonecan also be estimated simultaneously along with three morecompounds (glycerol, 3-hydroxypropionaldehyde and 1,3-propane-diol) via HPLC based method. These are the four key substancesformed in themetabolic pathway duringmicroorganism fermentationfor production of 1,3-propane diol from glycerol (Chen et al., 2007).
Fig. 6. Structuring and segregation of a mathematica
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8. Role of intracytoplasmic membrane in glycerol oxidation
Oxidation of glycerol occurs in case of G. oxydans through theaction of enzyme glycerol dehydrogenase. This enzyme is membranebound and the dihydroxyacetone synthesized remains extracellular. Ithas been found that at the end of exponential phase G. oxydansdifferentiates by forming dense regions at the end part of the cell,which can be observed through light microscope (Claus et al., 1975).Detailed observance through freeze-fracture-etched whole cells andthin sections through broken-cell envelopes of differentiated cellsrevealed that these cells contained polar accumulations of intracyto-plasmic membranes attached to plasma membrane which are notfound in undifferentiated exponentially growing cells (White andClaus, 1982).
When such differentiated cells were tested for the activity of theplasma membrane associated glycerol dehydrogenase, it was demon-strated that these differentiated cells containing intracytoplasmicmembranes were 100% more active than the undifferentiated cells. Ithas been suggested on the basis of these results that suchintracytoplasmic membranes are formed at the end of active celldivision due to continued plasma membrane synthesis.
9. Problems related to dihydroxyacetone production
It has been found that both substrate (glycerol) as well as product(dihydroxyacetone) causes inhibition of growth of G. oxydans duringfermentation.
9.1. Inhibition on G. oxydans caused by glycerol
Glycerol inhibits dihydroxyacetone production from G. oxydans byaffecting its biological activity. The comparison of the maximalproductivities and specific rates evaluated for initial concentrationsof 31, 51, 76, 95, and 129 g/l of substrate showed that glycerol exerts aninhibitory effect both on growth and dihydroxyacetone production. Itwas also observed that glycerol presence at higher concentration leadsto an increase in the time necessary for the cells to reach theirmaximal level of specific rates (Claret et al., 1992). As we can observe inTable 7 the yield of conversion of glycerol into biomass Yx/s varied in
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l model for the dihydroxyacetone fermentation.
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an inversely proportional way to the initial substrate content. Thisphenomenon implicates to the inhibition of growth by the increase ofglycerol concentration.
9.2. Inhibition on G. oxydans caused by dihydroxyacetone
When a comparison was carried out between the culturescontaining an increasing initial dihydroxyacetone concentration (0–100 g/l), it was observed that dihydroxyacetone also caused theinhibition of the fermentation process (Bauer et al., 2005; Claret et al.,1993). Dihydroxyacetone first affected the growth of bacteria (cellulardevelopment stopped when dihydroxyacetone concentration reached67 g/l) and later it also affected oxidation of glycerol (dihydroxyace-tone synthesis occurred only when concentration of dihydroxyace-tone in the culture medium was lower than 85 g/l).
Inhibition of product formation by dihydroxyacetone can be wellexplained by feedback inhibition kinetics. At much higher concentra-tions of dihydroxyacetone an irreversible destruction of G. oxydanscell occurred (Ohrem and Voβ, 1996b). This toxic effect of dihydrox-yacetone could be modeled by a death rate kinetic and introduction ofa damaged cell type. Dihydroxyacetone also inhibits the activity of thepentose cycle as can be measured via the CO2 evolution rate. Theinhibitory effect of dihydroxyacetone can be well understood in Fig. 6.
These problems of product and substrate inhibition have been ofgreat concern in dihydroxyacetone fermentation. Glycerol anddihydroxyacetone have different modes of inhibition (Claret et al.,1993) and it has been suggested that dihydroxyacetone inhibitoryeffect is more restricted to enzymes involved in bacterial growth andoxidation of glycerol whereas glycerol inhibition is more towards thecells (at the transport level). One of the solutions to solve suchproblems is to develop new fermentation procedures, which may leadto diminish effect of such inhibition phenomenon. On optimization offermentation conditions and also by trying different fermentationmodes for dihydroxyacetone production, it was found that in the caseof fed-batch culture, kinetic parameters were optimal and also ahigher amount of glycerol was converted as compared to that of batchculture (Bories et al., 1991). Although this method provided someadvantage as compared to that of batch fermentation, the severe effectof dihydroxyacetone could not be avoided if fermentation was carriedout for long durations. Thus, a more efficient fermentation methodwas needed for this purpose, which was possible by performingdihydroxyacetone production in two stages. In the first stage viable,durable, not product-inhibited culturewas provided and in the secondstage high final dihydroxyacetone concentrations were obtained. Theseparation of the two stages was possible due to the capability ofG. oxydans to perform growth-independent product formation. It wasrealized that this two-stage repeated fed-batch process was able toprovide reproducible long-term fermentations (Bauer et al., 2005).Similarly, fed-batch fermentation mode was found to be the best-adjusted system in the case of inhibition by the substrate, i.e., glycerol(Claret et al., 1992). These problems can also be solved byimmobilization of cells in a polymeric matrix, as discussed before.Another important problem associated with dihydroxyacetone fer-mentation is the problem of oxygen limitation. Currently availablemethods of improving oxygen limitation of dihydroxyacetone cultureshave also been discussed in detail in Section 6.
10. Conclusion and future prospects
Dihydroxyacetone has been found to have vast applications. It canbe recognized as a commercially important chemical. Dihydroxyace-tone production via microbial route has been found to be morefavorable as compared to that of chemical route. Therefore, most of theresearch conducted for dihydroxyacetone production is to enhancethe efficiency of microbial route of dihydroxyacetone production.Although, a lot of knowledge about fermentation for dihydroxyace-
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tone production has been known till now yet the desired yield (suchthat it meets the commercial requirement) has still not been achieved.According to the state of the arts, fermentation for dihydroxyacetoneproduction has been done via batch or fed-batch mode but thesemethods are not much promising due to inhibitory effect of glycerol aswell as dihydroxyacetone.
It has recently been reported that immobilization can be used fordihydroxyacetone production (Wei et al., 2007a). This method is stillin its infancy but it holds a great promise for the future study in thisfield because it has been reported that much higher yields ofdihydroxyacetone have been obtained by immobilization of G. oxydansand that too at a low production cost (Wei et al., 2007b). Thus, it can benoticed that significant advancements are being made in this field.Further optimizations of existing methods as well as development ofnovel methods based on existing knowledge about fermentation ofdihydroxyacetone will lead to successful results. Also new cloning andexpression vector have been constructed for G. oxydans (Schleyeret al., 2008; Shinjoh and Hoshino, 1995). These studies can be wellexploited for construction of vector systems expressing the gene ofinterest, i.e., glycerol dehydrogenase gene, at higher and cost effectivelevel. Thismethodwill possibly increase the yield of dihydroxyacetoneif in case the desired yield is not obtained by using native strain.
Acknowledgements
The work was financially supported from the grants from IndianInstitute of Technology, Kanpur, India and Department of Biotechnology(DBT), Department of Science and Technology (DST), Govt. of India.Ruchi Mishra gratefully acknowledges the fellowship received fromIndian Institute of Technology, Kanpur, India for her PhD program.
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