Recent advances in the biological production of mannitol - Springer Link

Appl Microbiol Biotechnol (2009) 84:55–62 DOI 10.1007/s00253-009-2086-5

MINI-REVIEW

Recent advances in the biological production of mannitol Seung Hoon Song & Claire Vieille

Received: 1 May 2009 / Revised: 5 June 2009 / Accepted: 6 June 2009 / Published online: 4 July 2009 # Springer-Verlag 2009

Abstract Mannitol is a fructose-derived, 6-carbon sugar alcohol that is widely found in bacteria, yeasts, fungi, and plants. Because of its desirable properties, mannitol has many applications in pharmaceutical products, in the food industry, and in medicine. The current mannitol chemical manufacturing process yields crystalline mannitol in yields below 20 mol% from 50% glucose/50% fructose syrups. Thus, microbial and enzymatic mannitol manufacturing methods have been actively investigated, in particular in the last 10 years. This review summarizes the most recent advances in biological mannitol production, including the development of bacterial-, yeast-, and enzyme-based transformations. Keywords Mannitol . Fructose . Lactic acid bacteria . Mannitol biological production . Cofactor regeneration . Glucose

Introduction Sugar alcohols are a class of polyols in which the sugar’s carbonyl (aldehyde or ketone) is reduced to the corresponding primary or secondary hydroxyl group. They are found naturally in fruits and vegetables, and they are produced by microorganisms (Akinterinwa et al. 2008). The six-carbon S. H. Song : C. Vieille (*) Department of Microbiology & Molecular Genetics, 2215 Biomedical Physical Sciences Building, Michigan State University, East Lansing, MI 48824-4320, USA e-mail: [email protected] C. Vieille Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA

polyol, D-mannitol, is the most abundant polyol in nature. It is produced by bacteria, yeasts, fungi, algae, lichens, and many plants (Wisselink et al. 2002). In many of these organisms, it is used as carbon and energy source, carbon storage (Pharr et al. 1995; Soetaert et al. 1999), and osmoregulatory compound in response to abiotic stress (Jennings 1984; Kets et al. 1996; Stoop et al. 1996). Mannitol is the main storage carbon in the mushroom, Agaricus bisporus, representing 50% of the fruit body dry weight (Pharr et al. 1995). In Aspergillus candidus, up to 50% of the glucose consumed can be converted to mannitol (Smiley et al. 1967). Because mannitol oxidation produces NAD(P)H that can be indirectly converted to ATP, mannitol is a highly efficient respiratory source, and mannitol metabolism is thought to be involved in growth regulation through its possible control of the cellular NADP/NADPH ratio (Stoop and Mooibroek 1998). Mannitol has several properties that make it an attractive food and pharmaceutical ingredient. It has glycemic and insulinemic indexes of 0, and it is not metabolizable in the body. Indeed, 75% of ingested mannitol gets fermented by the intestinal flora. The remaining 25% is absorbed before being excreted in urine (Livesey 2003). Because it is virtually noncariogenic, mannitol is used as a low-caloric and lowcariogenic sweetener, in particular in food for diabetic patients (Le and Mulderrig 2001). Its very low hygroscopicity makes it useful for products that require stability at high humidity. Because it does not react with active components in drugs and because its cool sweet taste masks the unpleasant taste of some drugs, it is often used as a pharmaceutical formulating agent (e.g., dental hygiene products and low reactivity drug fillers; Le and Mulderrig 2001; Soetaert et al. 1999). In medicine, mannitol is used as a diuretic in the manufacture of intravenous fluids. Finally, it is also used as a specialty chemical in other industrial applications (Soetaert et al. 1995).

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Appl Microbiol Biotechnol (2009) 84:55–62

SpecChem Online estimates “the global market for sorbitol, xylitol, and mannitol to be about €745 million, with sorbitol accounting for 83%, xylitol 14%, and mannitol 3%” (www.specchemonline.com, Sugaring the pill, October 21, 2004). While over 70% of the mannitol produced in China in 2005 was extracted as a by-product of alginate and iodine production from seaweed (Chinese Chemical Market, 2005), the rest of the world produces mannitol (50,000 tons/year) industrially by hydrogenation of 50% fructose/50% glucose syrups at high pressures (70– 140 atm) and high temperatures (120–160°C) using a Raney nickel catalyst and hydrogen gas (Kulbe et al. 1987; Soetaert et al. 1999). In this reaction, glucose is hydrogenated into D-sorbitol, while fructose is hydrogenated into a mixture of D-mannitol and D-sorbitol, yielding a 30% mannitol–70% sorbitol mixture. Mannitol is then purified by chromatography to remove the metal catalyst, followed by low-temperature crystallization to separate it from sorbitol (Soetaert et al. 1999). This chemical hydrogenation process has several limitations, including the need for highly purified substrates, high reaction temperatures and pressures, and a costly chromatographic purification step (von Weymarn et al. 2003), as well as a poor final mannitol yield (only ~15% crystalline mannitol). In this process, mannitol is only the side product in a reaction that produces mostly sorbitol, a situation that makes mannitol production depend on the market and price of sorbitol. Because of all the drawbacks associated with the current mannitol production process and because of mannitol’s increasing market, biological mannitol production processes are actively being investigated (Racine and Saha 2007; Silveira and Jonas 2002; Soetaert et al. 1999; von Weymarn et al. 2003). Jennings (1984) and Soetaert et al. (1999) reviewed the production of mannitol by fungi and osmophilic yeasts. Yields reached up to 50–52% on glucose or glycerol. This review will focus mostly on more recent advances in this field, including bacterial-, yeast-, and enzyme-based transformations.

Mannitol production by lactic acid bacteria As early as 1921 (Fred et al. 1921), lactic acid bacteria (LABs) were grouped into homofermentative species, which ferment lactose into lactic acid with only traces of other products, and heterofermentative species, whose fermentation balances on glucose and fructose follow Eqs. 1 and 2, respectively: 1 glucose ! 1 lactic acid þ 1 ethanol þ 1 CO2

ð1Þ

3 fructose ! 2 mannitol þ 1 lactic acid þ 1 acetic acid þ 1 CO2

ð2Þ

In contrast to heterofermentative species, homofermentative LABs do not naturally produce mannitol from fructose. Homo- and heterofermentative LABs are differentiated by their hexose uptake and utilization pathways (Wisselink et al. 2002). Both pathways will be briefly described in relation to mannitol production. Mannitol production by homofermentative LABs Homofermentative LABs ferment hexoses through glycolysis. Before entering glycolysis, sugars such as glucose and fructose are phosphorylated by the phosphoenolpyruvatedependent sugar/phosphotransferase system (PTS; Thompson 1987) when translocated into the cell. Fermentation of 1 mol of glucose by homofermentative LABs typically results in the formation of 2 mol lactic acid, 2 mol CO2, and 2 mol ATP (Fig. 1a). In homofermentative LABs, mannitol synthesis starts from fructose-6-phosphate. Mannitol-1phosphate dehydrogenase (M1P-DH, EC 1.1.1.17) reduces fructose-6-phosphate into mannitol-1-phosphate (Ferain et al. 1996), which in turn is dephosphorylated to mannitol by mannitol 1-phosphatase (M1Pase). Little if any M1P-DH activity is detected in hexose-grown Lactococcus lactis (Nyyssölä and Leisola 2005; Wisselink et al. 2002). While they do not naturally produce mannitol, homofermentative LABs are widely used in dairy fermentation. Engineering homofermentative LABs to produce mannitol could increase the viability of starter cultures and result in fermented dairy products with extra value. Two studies so far have focused on engineering L. lactis to produce mannitol (Gaspar et al. 2004; Wisselink et al. 2005). Inactivation of lactate dehydrogenase, the enzyme responsible for NAD+ regeneration in anaerobic conditions, seems to be a prerequisite for L. lactis to produce mannitol (Neves et al. 2000; Wisselink et al. 2004). Mannitol can be used as carbon source by L. lactis in the absence of glucose, and mannitol uptake takes place through the mannitol-specific PTS (PTSmtl; Gaspar et al. 2004). A mannitol-producing strain of L. lactis was obtained by deleting mtlF (encoding EIIAmtl in the PTSmtl) in a food grade Δldh L. lactis strain. Glucose metabolism by non-growing cells of the ΔldhΔmtlF strain produced mannitol as a major end product (Table 1; Gaspar et al. 2004). In a different approach, the Lactobacillus plantarum mtlD gene (encoding M1P-DH) and the Eimeria tenella mannitol 1-phosphatase (M1Pase) gene were coexpressed in a Δldh L. lactis strain. Optimizing induction conditions and substrate concentrations resulted in a glucoseto-mannitol conversion of up to 50 mol% (Wisselink et al. 2005). In both studies, formate, lactate, ethanol, and 2,3butanediol were also produced in significant quantities. Mannitol production by heterofermentative LABs Heterofermentative LABs, including Leuconostoc and Lactobacilli Group III (obligately heterofermentative), use the 6-

Appl Microbiol Biotechnol (2009) 84:55–62 Glucose glc

57

Fructose

Mannitol

frc

PTS

PTS

PTS

Glucose-6-P

Fructose-6-P

2

NADH

mannitol (Martinez et al. 1963; Sakai and Yamanaka 1968; Wisselink et al. 2002). Heterofermentative LABs do not express a M1P-DH, and their MDH is inactive on mannitol-1-phosphate, fructose-1-phosphate, and fructose6-phosphate (Grobben et al. 2001, Martinez et al. 1963). Reciprocally, no MDH activity has been detected in homofermentative LABs (Wisselink et al. 2002). Thus, homo- and heterofermentative LABs clearly differ in their mannitol production pathways. It was shown that when grown on glucose plus fructose, heterofermentative LABs preferentially use glucose as carbon source for metabolism and to produce ATP. In these growth conditions, fructose is reduced to mannitol to replenish the cell’s NAD+ pool. In these conditions, the fermentation follows Eq. 3 (Grobben et al. 2001):

mtl

Mannitol-1-P +

NAD

Glycolysis

Ethanol

(A)

Pyruvate 1

Acetate

Lactate

Fructose

Glucose

2 Fructose

Permease

Permease

Permease

Fructose 3

Glucose 4

2 Fructose

Fructose-6-P

2 Mannitol

7

NADH

Pyruvate 1

(B)

6 2 NAD+

NAD+

+

Ethanol

2 NADH

Xylulose-5-P 5

2 NADH 2 NAD

þ 1 acetic acid þ 1CO2

ð3Þ

Glucose-6-P

Pentose phosphate pathway

Acetate

1 glucose þ 2 fructose ! 2 mannitol þ 1 lactic acid

NADH +

NAD

Lactate

Fig. 1 Hexose metabolism in homofermentative (a) and heterofermentative LABs (b). PTSglc, PTSfrc, and PTSmtl: glucose-specific, fructose-specific, and mannitol-specific phosphotransferase systems, respectively; 1 lactate dehydrogenase, 2 mannitol-1-phosphate dehydrogenase, 3 fructokinase, 4 glucokinase, 5 phosphoketolase, 6 mannitol dehydrogenase, and 7 unknown. Solid arrows hexose assimilation pathways, dashed arrows fructose reduction to mannitol in glucose/fructose-grown heterofermentative LABs

phosphogluconate/phosphoketolase pathway for hexose fermentation (Kandler and Weiss 1986). In contrast to homofermentative LABs, heterofermentative species do not use the PTS for hexose uptake. Glucose and fructose enter the cell through permeases, before being phosphorylated by glucokinase and fructokinase, respectively (Fig. 1b). In heterofermentative LABs, mannitol is produced from fructose, rather than from fructose-6phosphate. Fructose reduction to mannitol is catalyzed by an NADH-linked mannitol dehydrogenase (MDH, EC 1.1.1.67), which reduces fructose exclusively to

Because the cells switch to making acetate (and one more ATP) rather than ethanol, mannitol-producing cultures grow faster than non-mannitol-producing ones (Wisselink et al. 2002). As can be seen from Eq. 3, even in a LAB-based mannitol bioconversion process using resting cells rather than growing cells, the maximum theoretical yield will be limited to 0.67 mol mannitol/mol total sugar. Most improvements of mannitol production by heterofermentative LABs have focused on optimizing mannitol conversion processes using wild-type strains. LABs are fastidious organisms that are unable to synthesize many of their essential building blocks, and LAB growth media typically use expensive yeast extract and peptone. Fructose is also a relatively expensive substrate compared to glucose. To produce mannitol cost-effectively on an industrial scale by fermentation, economical carbon and nitrogen sources are required to replace fructose syrup, peptone, and yeast extract. Saha investigated the effects of molasses, various inorganic, organic, and complex nitrogen sources and corn steep liquor on mannitol and lactic acid production by Lactobacillus intermedius NRRL B-3693. Soy peptone and corn steep liquor were found to adequately replace Bacto-peptone and Bacto-yeast extract in batch fermentations, and only complex nitrogen sources could support proper growth and mannitol production (Saha 2006a). L. intermedius was also shown to produce mannitol in high yields from inulin in a simultaneous saccharification and fermentation (SSF) process (Table 1; Saha 2006b). Inulin is a polymer of β-2,1-linked fructose with a glucose residue at its reducing end and found as a storage polymer in a variety of tubers and roots. In optimized conditions (with a mix of

58


Table 1 Characteristics of biological mannitol production processes Process characteristics

Substrate

Yielda

L. lactis, Δldh ΔmtlF

Resting cells

Glc 7.2 g/l

32.8

L. lactis Δldh, overexpressing M1PDH and M1Pase L. pseudomesenteroides, fructokinase inactivation L. mesenteroides

Growing cells

Glc 20 g/l

50

Growing cells

Glc 10 g/l and frc 20 g/l

Batch ferm. Fed-batch ferm.

Glc 50 g/l frc 100 g/l Glc/frc ratio of 1:2

Organism

r (g/h/l)

Final mannitol concentration (g/l)

References

2.7

2.4

(Gaspar et al. 2004)

0.16

9

(Wisselink et al. 2005)

53.7 (85.7)

2.8

NA

(Helanto et al. 2005)

60.7 (91) NA

3.75 6.3

90 150

(Soetaert et al. 1995)

Bacteria

L. mesenteroides M204

Fed-batch ferm.

Glc/frc ratio of 1:2

NA

L. mesenteroides

Resting cells in MCRB

Glc 50 g/l frc 100 g/l

64.7 (97)

26.2

98

(von Weymarn et al. 2002b)

L. mesenteroides

Resting cells in MCRB


58 (87)

18.8

87

(von Weymarn et al. 2003)

L. mesenteroides

Batch ferm.

37.6 (66.2)

1.8

18

(Fontes et al. 2009)

L. intermedius

Batch SSF

Cashew apple juice (Glc/frc ratio of 1:2) Inulin 300 g/l Frc 150 g/l and inulin 250 g/l

69b 57b

2.88 2.1

207 227

(Saha 2006b; 2006a)

L. intermedius

Fed-batch ferm. MCRB

Frc 67 g/l and glc 33.5 g/l Frc 100 g/l and glc 50 g/l

63 (94.6) 62 (93)

5.9 28.4

176 95

(Racine and Saha 2007)

L. fermentum

Batch ferm.


58.4 (89.6)

7.6

83

(von Weymarn et al. 2002a)

Resting cells

Frc 90 g/l and Na formate 17 g/l

84

8.2

66

(Kaup et al. 2004)

Resting cells

Glc 180 g/l and Na formate 34 g/l

80

3.6

146

(Kaup et al. 2005)

Fed-batch reactor with resting cells


91

2.97

285

(Bäumchen and Bringer-Meyer 2007)

Resting cells


91

0.92

22

(Bäumchen et al. 2007)

E. coli overexpressing MDH, FDH and GLF E. coli overexpressing MDH, FDH and GLF, plus extracellular XI C. glutamicum overexpressing MDH, FDH and GLF B. megaterium overexpressing MDH and FDH Yeasts

4.56

185

C. magnoliae HH-01

Fed-batch ferm.

Frc 250 g/l and glc 50 g/l

70 (84)

1.94

213

(Lee et al. 2003)

C. magnoliae HH-01

Batch ferm. Ca2+ and Cu2+ added

Frc 250 g/l and glc 30 g/l

78.8 (88)

1.72

223

(Lee et al. 2007)

r volumetric productivity, NA not available, Frc fructose, Glc glucose, FDH formate dehydrogenase, GLF glucose facilitator, MDH mannitol dehydrogenase, XI xylose isomerase, MCRB membrane cell-recycle bioreactor a

Yields are in mole percent of total sugar. Values in parentheses are in mole percent of fructose

b

Yields in gram percent of total sugar

endo- and exo-inulinases and at 37°C), the SSF process yielded final mannitol concentrations over 200 g/l (Saha 2006b). Cashew apple juice is a cheap and abundant agriculture by-product of cashew nuts production that was also investigated as a feedstock for mannitol production with Leuconostoc mesenteroides. The first batch fermentation results (Table 1) suggest that this carbon source, which naturally contains ~55% fructose and ~45% glucose, is a

suitable feedstock for mannitol production as well (Fontes et al. 2009). Another approach to decreasing the cost of LAB-based mannitol production processes is to grow the bacterial biomass first, then repeatedly use the resting cells in membrane cellrecycle type bioreactors (MCRB; von Weymarn et al. 2002b). Von Weymarn et al. (2002b) studied the effects of biomass concentration, initial fructose concentration, and glucose-to-


fructose ratio on mannitol productivity and conversion rates. They obtained a stable, high-level production of mannitol in 14 successive bioconversion batches (von Weymarn et al. 2002b). Cell viability did not decrease between batches 1 and 10, the mannitol yield in batch 7 (98.7 mol/mol%) was as high as for single-batch conversions, and the concentrations of by-products lactate and acetate did not vary from batch to batch. Volumetric productivity increased significantly from fermentations with growing cells to an MCRB process with resting cells (Table 1). The scalability of this MCRB mannitol production process was tested at a 100-l pilot plant scale (von Weymarn et al. 2003). Even larger increases in volumetric productivity (up to 40 g/l/h, Table 1) were obtained with L. intermedius in a continuous cell-recycle fermentation process, in which continuous sugar syrup feeding was used to overcome limitations caused by high substrate concentrations (Racine and Saha 2007). While bioconversion processes using mixed feeds of one third glucose and two thirds fructose have produced high mannitol yields from fructose in general, they still suffer from the possible leakage of fructose to the phosphoketolase pathway (von Weymarn et al. 2002a). One recent study focused on improving mannitol yields by isolating a mutant derivative of Leuconostoc pseudomesenteroides lacking fructokinase activity. Mutant BPT143 showed only 10% of the fructokinase activity measured in the wild-type strain. Strain BPT143 grew and consumed fructose faster than the parent strain when grown a glucose–fructose mix, and reduced leakage of fructose into the phosphoketolase pathway resulted in an increased mannitol yield from fructose (from 74 to 86 mol/mol; Helanto et al. 2005).

59 Glucose 1

Glucose GLF Glucose

Fructose GLF Fructose GLF

2 Fructose

NADH 3

Mannitol

Mannitol

CO2

CO2

4 NAD

+

Formate

Formate

Fig. 2 Mannitol production by E. coli from fructose or glucose using formate dehydrogenase to regenerate NADH. 1 Extracellular xylose isomerase, 2 co-expressed intracellular xylose isomerase; 3 MDH, 4 FDH

providing the enzyme extracellularly (Fig. 2) led to a less efficient conversion of glucose to mannitol with only 420 mM mannitol produced from 1,000 mM glucose (Kaup et al. 2005). Because E. coli showed poor stability in the presence of high concentrations of formate in the bioconversion, which led to decreases in overall productivity and to the inability to reuse the biomass for repeated bioconversions, a similar approach was repeated using Bacillus megaterium and Corynebacterium glutamicum expressing MDH and FDH. The processes catalyzed by B. megaterium and C. glutamicum had mannitol yields over 90 mol% from fructose with no accumulation of by-products (Table 1), and the biomass could be used in repeated bioconversions. The volumetric productivity was low, though, likely due to a slow fructose uptake rate (Bäumchen and Bringer-Meyer 2007; Bäumchen et al. 2007).

Mannitol production by other bacterial species Mannitol production in yeast A whole-cell biotransformation system for the production of mannitol from fructose was developed in Escherichia coli. The recombinant strain co-expressed the L. pseudomesenteroides mdh gene, encoding MDH; the Mycobacterium vaccae fdh gene, encoding formate dehydrogenase (FDH); and the Zymomonas mobilis glf gene, encoding the glucose facilitator protein (GLF). The recombinant strain was able to take up fructose independently from the PTS and to stoichiometrically couple fructose reduction to mannitol with formate oxidation to CO2 (Fig. 2). This bioconversion produced mannitol with a good volumetric productivity (Table 1) and essentially no byproduct (Kaup et al. 2004). Supplementing the recombinant strain expressing MDH, FDH, and GLF with extracellular xylose isomerase resulted in the formation of 800 mM D-mannitol from 1,000 mM D-glucose (Fig. 2, Table 1). Overexpressing xylose isomerase intracellularly rather than

Candida magnoliae HH-01 was isolated from fermentation sludge during a screening of over 1,000 microorganisms for mannitol production. C. magnoliae produced up to 208 g/l mannitol from fructose in a fed-batch fermentation (Song et al. 2002). Improvements of the fed-batch conversion process have since included co-feeding glucose with fructose and supplementing the culture with Ca2+ and Cu2+ (Table 1; Baek et al. 2003; Lee et al. 2003; Lee et al. 2007). In C. magnoliae, mannitol is produced from fructose by an NADP-dependent mannitol dehydrogenase. The glucose co-feed, rather than fructose, is used for cell maintenance and NADPH regeneration (Lee et al. 2003). Lee et al. (2007) suggested that Ca2+ increases cell permeability and mannitol secretion, while Cu2+ increases the activity of MDH.

60


(A) Fructose

Gluconic acid

NADH 1

2 NAD+

Mannitol

Glucose

(B) Fructose

NADH 3

Mannitol

Expressing mtlD could not restore anaerobic growth to the double mutant strain, though (Costenoble et al. 2003).

CO2 4

NAD+

Formate

Fig. 3 Enzymatic production of mannitol from fructose with cofactor regeneration. a 1 S. cerevisiae MDH; 2 B. megaterium glucose dehydrogenase (Kulbe et al. 1987); b 3 Pseudomonas fluorescens MDH; 4 C. boidinii FDH (Slatner et al. 1998)

While the volumetric productivity of C. magnoliae processes remains low, the final mannitol concentrations are among the highest reported (Table 1). C. magnoliae is reported to produce little by-products during mannitol production, but more information is not available. Rather than increasing or remaining stable, the yeast biomass decreases significantly during the >100-h fed-batch process (Lee et al. 2003; Lee et al. 2007). Whether this biomass decrease might affect process sustainability remains in question. Mannitol production has been attempted with Saccharomyces cerevisiae. S. cerevisiae does not naturally produce mannitol. Under anaerobic conditions, S. cerevisiae regenerates NAD+ by producing glycerol. A Δgpd1 Δgpd2 double mutant, devoid of NADH-dependent glycerol-3-phosphate dehydrogenase activity (Gpd1p and Gpd2p), is unable to grow anaerobically. Expression of E. coli mtlD, encoding Mt1P-DH, in the S. cerevisiae double mutant resulted in NADH oxidation by Mt1P-DH and in 12 mol% mannitol production from glucose.

Development of an enzyme-catalyzed mannitol production process Enzyme-based cofactor regeneration Instead of using whole cells, Kulbe et al. (1987) combined purified enzymes in a single membrane reactor to produce mannitol and gluconic acid from a glucose–fructose syrup. In this process, the NADH consumed in mannitol production by S. cerevisiae MDH was regenerated during gluconic acid production by B. megaterium glucose dehydrogenase (Fig. 3a). They obtained turnover numbers over 100,000 for NADH regeneration and 88% fructose conversion to mannitol. Limiting factors in the process included poor enzyme stability and substrate inhibition (Kulbe et al. 1987). To remove the downstream separation of mannitol away from gluconic acid, mannitol production was also tested with Candida boidinii formate dehydrogenase as the second enzyme (Fig. 3b). In this system, the second product, CO2, is easily removed from the reaction solution, and it does not inhibit the MDH reaction. In the batch reactor, the productivity was 2.2 g/l/h, with ~1,000 cofactor turnovers (Slatner et al. 1998). A similar conversion was performed using the L. mesenteroides MDH and C. boidinii FDH that reached over 95 mol% conversion after 70 h (Parmentier et al. 2003). Electrochemical cofactor regeneration Today, the only industrially used cofactor regeneration system is the use of C. boidinii FDH in L-tert-leucine production (Bommarius et al. 1998). Low specific activity, production cost, and formate cost and low stability in organic solvents (van der Donk and Zhao 2003) restrict the use of FDH to the production of high-value chemicals, though. A recently

Table 2 Comparison of the catalytic hydrogenation process with microbial and enzymatic conversion methods Factor

Catalytic hydrogenation

Biological conversion

Catalyst Substrate(s) Process conditions Mannitol Theoretical yield from fructose Theoretical yield from total sugar

Non-specific High purity needed High pressure and high temperature Side product 50% 25%

Other products and impurities

Sorbitol and Ni catalyst, both difficult to separate ≤39% mol/mol initial sugar

Specific Low-purity can be used 30–37°C, 1 atm Main product 100% 67% (LAB-based fermentation), 100% (resting cells or enzymatic) Organic acids, ethanol, and sugars, all easy to separate

Yield of crystalline mannitol Adapted from von Weymarn et al. (2003)

52% mol/mol initial sugar


discovered bacterial phosphite dehydrogenase could be a convenient alternative to FDH. Phosphite and phosphate are innocuous to most enzymes, and they would not interfere with product recovery (Vrtis et al. 2002). This enzyme remains to be tested in large-scale settings, and its specific activity is as low as that of FDH (van der Donk and Zhao 2003). Another approach to cofactor regeneration is electrochemical regeneration. Electrochemical recycling of pyridine nucleotide cofactors is a promising technology that has already been extensively studied. Many of the potential obstacles (e.g., electrode fouling, cofactor dimerization, and the necessity of high overpotentials) have been overcome through method development. In this approach, redox enzyme and cofactor are immobilized on the cathode of a bioelectrochemical reactor, and electricity provides the electrons necessary to reduce the cofactor (Hassler et al. 2007). Such system can be developed, provided that a robust MDH is available. In preliminary experiments, thermostable Thermotoga maritima MDH (Song et al. 2008) and thermostable Thermotoga neapolitana xylose isomerase mutant 1F1 (Sriprapundh et al. 2003), both highly active at pH 6.0, have been co-immobilized on an electrode together with NADH and an electron mediator. This enzyme– electrode interface produced 180 mM mannitol from 300 mM glucose in the absence of fructose accumulation (Hassler et al. manuscript in preparation). In conclusion, even though biological processes for mannitol production are still being optimized, they already compare very favorably with the current Ni-catalyzed hydrogenation process (Table 2). Not only does mannitol become the main product in biological conversions but it can be also produced using feedstock cheaper than fructose. According to a 2005 commercialization announcement (http:// www.foodnavigator-usa.com/Financial-Industry/ZuChemgears-up-for-first-mannitol-sweetener), the American company zuChem Inc. is close to commercializing mannitol production by L. intermedius NRRL B-3693. While the LAB-based bioconversion processes have organic acids, ethanol, and sugars as by-products—which are all easy to separate from mannitol—whole-cell transformations (such as that tested with E. coli and B. megaterium) or enzymatic conversions have the potential to produce mannitol essentially without by-products. More research is needed to be able to predict whether whole-cell transformations or enzymatic conversions can also become commercially viable. Chinese consumption of mannitol increased by more than 23% per year since year 2000, mostly for use in pharmaceutical injections (Chinese Chemical Market, 2007). If the evolution of the Chinese demand for mannitol reflects trends in the world market, the incentive is there to increase the yield and volumetric productivity of biological

61

mannitol manufacturing processes and bring these processes to market.

Acknowledgment This work was supported by the National Research Initiative grant number 2008-35504-04611 from the United States Department of Agriculture’s Cooperative State Research, Education, and Extension Service.

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