Fumaric acid production by fermentation

The potential of fumaric acid as a raw material in the polymer industry and the increment of cost of petroleum-based fumaric acid raises interest in fermentation processes for production of this compound from renewable resources. Although the chemical process yields 112% w/w fumaric acid from maleic anhydride and the fermentation process yields only 85% w/w from glucose, the latter raw material is three times cheaper. Besides, the fermentation fixes CO2. Production of fumaric acid by Rhizopus species and the involved metabolic pathways are reviewed. Submerged fermentation systems coupled with product recovery techniques seem to have achieved economically attractive yields and productivities. Future prospects for improvement of fumaric acid production include metabolic engineering approaches to achieve low pH fermentations.
Introduction
Fumaric acid (Fig. 1) is a naturally occurring organic acid. It was first isolated from the plant Fumaria officinalis, from which it derives its name. Many microorganisms produce fumaric acid in small amounts, as it is a key intermediate in the citrate cycle. Fumaric acid is also known as (E)-2-butenedioic acid or trans-1,2-ethylenedicarboxylic acid. Sometimes the term “fumarates” is also used. In this review, this general term is not used to describe fumaric acid esters like dimethyl fumarate but only to describe the salts of fumaric acid (sodium fumarate, calcium fumarate).
Fig. 1Current applications of fumaric acid production. Miscellaneous include: lubricating oil, inks, lacquers, carboxylating agent for styrenebutadiene rubber, personal care additives. a Anonymous 2007; b Willke and Vorlop 2004
Currently, fumaric acid is produced chemically from maleic anhydride, which in turn is produced from butane. However, as petroleum prices are rising rather quickly, maleic anhydride as a petroleum derivative has increased in price as well (Anonymous 2007). This situation has caused a renewed interest in the fumaric acid production by fermentation that was operational during the 1940s (Goldberg et al. 2006), but was discontinued and replaced by petrochemical processes. The fermentation process is also interesting because it involves carbon dioxide fixation, as will be discussed later. Fungi are known for their organic acid-producing capability and have been used in fermentation processes for fumaric acid production.
Production by filamentous fungi of organic acids, including fumaric acid, has recently been reviewed (Magnuson and Lasure 2004; Goldberg et al. 2006). These reviews focused on the microorganisms with their associated metabolic pathways. In contrast, the current review focuses on development of fumaric acid production processes. After summarizing fumaric acid properties, applications, and production, metabolic pathways for fumaric acid production will be discussed only briefly before turning to fermentation performance of the most prominent fumaric acid-producing strains. Furthermore, different methods that have been studied to optimize fermentation processes will be mentioned, and future prospects for process development will be discussed.
Properties and applications of fumaric acid
Because of its structure (a carbon–carbon double bond and two carboxylic acid groups), fumaric acid has many potential industrial applications (Fig. 1). It can act as starting material for polymerization and esterification reactions (www.the-innovation-group.com/ChemProfiles/Fumaric%20Acid.htm; 10/06/07).
As raw material for polymerization, especially in the production of unsaturated polyester resins, maleic anhydride is currently preferred to fumaric acid because maleic anhydride [1.46–1.63 $/kg; (Anonymous 2007)] is cheaper than fumaric acid, the latter historically being around 10% more expensive than maleic anhydride (http://www.chemweek.com/inc/articles/t/2007/04/04/04/022.html). However, fumaric acid could be a better option for the polymer industry among other carboxylic acids or their derivatives because of its nontoxic nature. In addition, special properties like greater hardness in the polymer structure can be achieved when fumaric acid is used (www.the-innovation-group.com/ChemProfiles/Fumaric%20Acid.htm; 10/06/07). In addition to polymerization, there are two potentially new applications for fumaric acid and both require a different grade of purity of it. The first is as a medicine to treat psoriasis, a skin condition (Altmeyer et al. 1994; Mrowietz et al. 1998). Psoriatic individuals are unable to produce fumaric acid in their body (which is not the case in normal individuals) due to a certain biochemical defect that interferes with adequate fumaric acid production in the skin. Therefore, psoriatic individuals need to take orally fumaric acid in the form of fumaric acid monoethyl or dimethyl ester to treat their disease. The second potential application of fumaric acid is as supplement in cattle feed. Recent studies indicate that a large reduction in the methane emissions of cattle can be achieved (up to 70%), if this cattle receives fumaric acid-based additive as a supplement in their diet (Mcginn et al. 2004). This could greatly reduce the total emission of methane, as farm animals are responsible for 14% of the methane emission caused by human activity.
Fumaric acid, together with the related succinic and malic acids, has been identified as one of the top ten building block chemicals that can be produced from sugars via biological or chemical conversion (Werpy and Petersen 2004). Compared to these other dicarboxylic acids, fumaric acid has a low aqueous solubility [7 g/kg at 25°C; 89 g/kg at 100°C (Stephen 1965)] and low pKa values [3.03 and 4.44 (Lohbeck et al. 1990)], which are properties that can be exploited for product recovery.
Fumaric acid production by petrochemical routes
Fumaric acid is currently produced by isomerization of maleic acid, which is produced from maleic anhydride. Maleic anhydride, in turn, is industrially produced by catalytic oxidation of suitable hydrocarbons in the gas phase. Benzene used to be the predominant starting material, but oxidation of n-butane or n-butane–n-butene mixtures has become more important in recent years (Lohbeck et al. 1990). The butane oxidation reaction equation to maleic anhydride is: C4H10 + 3.5O2 → C4H2O3 + 4H2O.
In the most common maleic anhydride process, the catalyst is embedded in fixed-bed tubular reactors. The catalysts applied in this process are based on vanadium and phosphorus oxides. In this process, water is formed as a by-product, and a fairly small amount of it can be directly liquefied from the reaction gas by partial condensation. Organic solvents absorb maleic anhydride contained in the reaction gas. More than 98% of the anhydride can be absorbed in this way. The solvent–anhydride mixture is then subjected to fractional distillation to separate maleic anhydride from the solvent, and the latter is returned to the absorption column (Lohbeck et al. 1990). The thus obtained pure maleic anhydride is then hydrolyzed into maleic acid according to the reaction equation C4H2O3+H2O → C4H4O4.
Subsequently, the maleic acid is converted almost quantitatively by thermal or catalytic cis–trans isomerization into fumaric acid (Lohbeck et al. 1990). The most common catalysts used are mineral acids, peroxy compounds, and thiourea. The crude fumaric acid obtained in this way is purified by crystallization from water (Fig. 2). As will be shown later, a comparable purification may be used for fermentative fumaric acid production.
Fig. 2Plant for fumaric acid production from malic acid. a Isomerization vessel; b Centrifuge; c Dissolving tank; d Filter; e Crystallizer; f Dryer (adapted from Felthouse et al. 2001)
Production capacity of maleic anhydride in 2007 was about 1,807,000 ton/a (Anonymous 2007), with around 3% of this being used for fumaric acid production (http://chemicalmarketreporter.com/home/Default.asp?type = 17&liArticleID = 2015643...; 18/04/2007), corresponding to 90,000 ton/a.
Enzymatic conversion of maleic acid into fumaric acid
In the aforementioned chemical isomerization of maleic acid into fumaric acid, the conversion yield is restricted by the reaction equilibrium. This chemical conversion is occurring at high temperatures that causes formation of by-products from maleic and fumaric acids, and as a consequence, yields are below the equilibrium yields (Goto et al. 1998). This problem was the main motivation to look for a good enzyme that would facilitate isomerization, so that the equilibrium mixture would be obtained without formation of by-products.
It is known that the enzyme maleate isomerase (maleate cis-trans-isomerase) isomerizes maleic acid into fumaric acid. Microorganisms that produce maleate isomerase are Pseudomonas species (Otsuka 1961), Alcaligenes faecalis (Takamura et al. 1969), and Pseudomonas fluorescens (Scher and Lennarz 1969). However, maleate isomerase is unstable even at moderate temperatures (Takamura et al. 1969), and therefore, a heat-stable maleate isomerase is desired. Thermo-stable maleate isomerases derived from Bacillus stearothermophilus, Bacillus brevis, and Bacillus sp. MI-105 were reported by Goto et al. (1998), and according to the authors, the use of enzymes from these bacteria can improve the fumaric acid production process.
Furthermore, in a more recent study using Pseudomonas alcaligenes XD-1, high rates of conversion of maleic acid into fumaric acid (6.98 g L−1 h−1) were reported (NakajimaKambe et al. 1997). This organism does not normally accumulate fumaric acid, but by heat treatment of the cells (70°C for 1 h), they lost their fumarase activity, which otherwise would be responsible for a conversion of fumaric acid into l-malic acid. This heat treatment did not affect the maleate isomerase activity. In addition, when calcium ions were added during the conversion step, the thermal stability of maleate isomerase was increased. With the use of P. alcaligenes XD-1, the highest reported conversion yield, 95%, was achieved in the conversion of maleic acid to fumaric acid (Ichikawa et al. 2003). Immobilization of the heat-treated cells is currently under investigation.
Microbial production of fumaric acid
Fumaric acid production by fermentation was operated in the United States during the 1940s, but later, this process was discontinued and replaced by chemical synthesis from petrochemical feedstocks, the latter being explained in a previous section. Nevertheless, the continuous increase of the petroleum prices has brought back the interest in fumaric acid production by submerged fermentation (Goldberg et al. 2006). Fumaric acid production by fermentation using Rhizopus species has been patented occasionally (Waksman 1943; Kane 1943; Lubowitz and La Roe 1958; La Roe 1959; Goldberg and Stieglitz 1986). In 1989, DuPont patented an improved fermentation process producing carboxylic acids (fumaric, succinic, malic, and mixtures thereof) by controlling dissolved oxygen levels between 30 and 80% (Ling and Ng 1989).
In the field of fumaric acid production by fermentation, there are many aspects determining the productivity of the fermentation process, such as the applied microbial strain and its morphology, the use of a neutralizing agent, and the applied feedstock. Those aspects are reviewed and analyzed in more detail in the following sections.
Fumaric acid-producing strains
After the discovery of fumaric acid production in Rhizopus nigricans by Felix Ehrlich in 1911, Foster and Waksman (1938) screened 41 strains from eight different genera to identify high fumarate producing strains. The fumarate-producing genera identified were Rhizopus, Mucor, Cunninghamella, and Circinella species. Among these strains, Rhizopus species (nigricans, arrhizus, oryzae, and formosa) were the best-producing ones, yielding fumaric acid under aerobic and anaerobic conditions (Foster and Waksman 1938; Rhodes et al 1959; Kenealy et al. 1986; Cao et al. 1996; Carta et al. 1999). Table 1 lists these fungi, and as is shown, the R. arrhizus NRRL 2582 and R. oryzae ATCC 20344 gave the highest volumetric productivity, product titer, and product yield values (Gangl et al. 1990; Cao et al. 1996).
Table 1Literature data on fumaric acid production by different Rhizopus generaStrainFermenterSubstrateProduct Titer (g l−1)Yield (g g−1)Vol. Prod. (g l−1 h−1)Time (h)Final pHReferenceR. nigricans 45Shake flaskGlucose14.70.50–1686.5Foster & Waksman 1938Shake flaskGlucose20.00.660.25806.5Romano et al. 1967R. arrhizus NRRL 2582Stirred tankGlucose90.00.701.22726.0Rhodes et al. 1962Stirred tankGlucose107.00.822.00536.0Ng et al. 1986Stirred tankGlucose73.00.720.501475.5Gangl et al. 1990R. arrhizus NRRL 1526Shake flaskGlucose97.70.811.02966.0Kenealy et al. 1986Fluidized bedMolasses17.50.360.36486.0Petruccioli et al. 1996Stirred tankGlucose38.00.330.46825.5Riscaldati et al. 2000R. oryzae ATCC 20344RBCa plus AdsorptionGlucose92.00.854.25244.5Cao et al. 1996Stirred tankGlucose65.00.650.90725.0Cao et al. 1996RBCaGlucose75.50.753.78245.0Cao et al. 199710-l air liftGlucose37.80.750.81465.0Du et al. 1997Stirred tankGlucose35.80.600.90405.5Zhou 1999Bubble columnGlucose37.20.531.03365.0Zhou et al. 2002R. formosa MUCL 28422Stirred tankCassava bagasse21.3–––6.5Carta et al. 1999aRotatory biofilm contactor
Despite the fact that the aforementioned experimental studies involve fungi, the use of bacteria has been considered as well. Donnelly et al. (2001) have suggested using a Lactobacillus host strain that lacks the malolactate enzyme, fumarase, and fumarate dehydrogenase. Introducing the maeA gene for the malic enzyme from Escherichia coli would result in a pathway from pyruvate to malate and hence in a malic acid-producing mutant. Additional genetic engineering would result in fumaric acid production.
Genetic modification of microorganisms has hardly been explored for fumaric acid production but offers a potentially useful approach solution for improving yields and rates in fermentation.
Metabolic pathways to fumaric acid
Fumaric acid is primarily an intermediate of the citrate cycle, but is also involved in other metabolic pathways. In 1939, it was suggested that fumarate biosynthesis involved a C-2 plus C-2 condensation in Rhizopus species (Foster et al. 1949). The reactions in this pathway seemed to be similar to those of the glyoxylate bypass (Foster et al. 1949). Years after, the glyoxylate bypass mechanism was ruled out because the theoretical molar yield for this pathway of 100% was not in agreement with the experimental yield of 140% (Rhodes et al. 1959; Romano et al. 1967). However, the main evidence for rejecting the glyoxylate bypass mechanism is that the key enzyme of the glyoxylate pathway, isocitrate-glyoxylate lyase, was repressed when high glucose concentrations were present like in the experiments used for fumaric acid production (Romano et al. 1967).
It was discovered that a C3 plus C1 mechanism involving CO2 fixation catalyzed by pyruvate carboxylase under aerobic conditions explained the high molar yields in fumarate production (Overman and Romano 1969). This CO2 fixation leads to oxaloacetic acid formation (Osmani and Scrutton 1985), so that C4 citrate cycle intermediates can be withdrawn for biosynthesis during the growth phase under aerobic conditions. When nitrogen becomes limiting and the growth phase stops, the metabolism of glucose and CO2 fixation could continue and lead to an accumulation of C4 acids (Romano et al. 1967). The maximal theoretical yield in a nongrowth situation is 2 mol of fumaric acid per mole of glucose consumed, upon fixation of 2 mol of CO2 via reductive pyruvate carboxylation. However, if the reductive pyruvate carboxylation would be the sole pathway, no ATP would be produced for maintenance or transport purposes. Therefore, the citrate cycle is also active during fumaric acid production (Rhodes et al. 1959; Kenealy et al. 1986).
The CO2 carboxylation enzyme, pyruvate carboxylase, is known to be localized exclusively in the cytosol, together with NAD-malate dehydrogenase and fumarase (that are present in the cytosol and in the mitochondria), leading to fumaric acid synthesis in this cell compartment (Osmani and Scrutton 1985). Studies performed by Peleg et al. (1989) indicated higher activities of these enzymes (especially the cytosolic isoenzymes) during fumaric acid production. Kenealy et al. (1986) used mitochondrial inhibitors to investigate their role in fumarate accumulation and found no direct involvement of such inhibitors of the citrate cycle in fumarate production. However, carbon-labeling studies have demonstrated the simultaneous utilization of both the citrate cycle and the reductive pyruvate carboxylation pathways under aerobic conditions (Fig. 3). Besides the localization of fumarase isoenzymes, it was also found that addition of cycloheximide virtually eliminated the cytosolic isoenzyme and therefore caused a large decrease in the amount of fumaric acid produced by R. oryzae (Peleg et al. 1989).
Fig. 3Citrate cycle pathway and reductive carboxylation pathway leading to fumaric acid accumulation (adapted from Kenealy et al. 1986). The ratio between the two pathways is not 1:1 as suggested by this figure
The carboxylation of pyruvate has been studied in more detail for the analogous microbial succinate production, but the understanding of the different metabolic fluxes involved is still incomplete (McKinlay et al. 2007).
High-producing fumaric acid strains of Rhizopus not only produce fumaric acid but also other carboxylic acids like malic, lactic, acetic, succinic, and citric in smaller amounts than fumaric acid production (Rhodes et al. 1959; Carta et al. 1999). Sometimes, ethanol is also produced, like in the case of the high-producing strain R. oryzae ATCC 20344 (Cao et al. 1996). However, it was found that ethanol production can be reduced by a sufficient supply of oxygen to the culture (Cao et al. 1996).
Fumaric acid transport across the cell membrane
The transport mechanism of fumaric acid in fungi has not been studied yet. However, transport of l-malic acid and other dicarboxylic acids, including fumaric acid, has been studied in yeasts like Schizosaccharomyces pombe, Candida utilis, Candida sphaerica, and Hansenula anomala (Corte-Real et al. 1989; Corte-Real and Leao 1990; Saayman et al. 2000). These microorganisms may be comparable to fumaric-producing fungi. These studies showed that the initial uptake of malic acid was accompanied by disappearance of extracellular protons suggesting that the anionic form of the acid was transported by an accumulative dicarboxylate proton symporter. As fumaric acid seems to be a competitive inhibitor of l-malic acid uptake, it was suggested that fumaric acid uses the same import system.
On the other hand, these studies showed that undissociated dicarboxylic acid entered the cells slowly by simple diffusion. In addition, it was revealed that the rate of diffusion of the undissociated acid across the plasma membrane is subjected to opposite pH influences: an increase due to the relative increase of undissociated acid with decreasing pH and a decrease due to a decrease of the permeability of the cell membrane for the undissociated acid at decreasing pH (Corte-Real and Leao 1990). Increasing the number or activity of the dicarboxylic acid transporters could lower the intracellular fumarate concentration and could therefore have a positive effect on the production yield (van Maris et al. 2004).
Alternative fermentation feedstocks
Glucose has not been the only carbon source used for fumaric acid-producing strains. The use of xylose with immobilized R. arrhizus was studied, but the highest obtained volumetric productivity was only 0.087 g l−1 h−1 (Kautola and Linko 1989). Also, sucrose has been considered as a raw material in fumaric acid-production processes (Kautola and Linko 1989; Zhang et al. 2007), but sucrose has the disadvantage of being poorly metabolized by R. oryzae compared to glucose (Bulut et al. 2004).
Starch-containing materials were also screened as feedstock for the fumaric acid production. Potato flour was used as feedstock for R. arrhizus, and although fumaric acid was the main metabolic product, a volumetric productivity of only 0.35 g l−1 h−1 was achieved (Moresi et al. 1991). On the other hand, it was found that the highest yields of fumaric acid were obtained from acid hydrolysates of starch-based materials (Moresi et al. 2002). The same feedstock was used by Federici et al. (1993) in studies on the effect of agitation speed and applied neutralizing agent. The tested neutralizing agent was a combination of CaCO3 and KOH/KCO3, and it was concluded that comparable fumaric acid yields were obtained using these neutralizing agents and starch-based materials (Federici et al. 1993). Carta et al. (1999) optimized the fumaric acid production by R. formosa MUCL 28422 by using enzymatic hydrolysates of cassava bagasse, a high-starch-containing waste product. The productivity was not improved with respect to a fermentation where glucose was used as a feedstock, but the fact that these feedstocks are cheap and abundant makes these optimized fermentations economically attractive.
Nutrient requirements
Physical and nutritional requirements for fumaric acid fermentation have been studied in Rhizopus arrhizus (Rhodes et al. 1959; Rhodes et al. 1962). As Rhizopus species should enter a phase of limited growth during the fermentation, which can be achieved by nitrogen limitation, these studies showed that to achieve high yields during fumaric acid fermentation, the most critical parameter is the ratio of glucose to nitrogen. For example, a yield of fumaric acid on glucose of 85% (w/w) was obtained using an initial C:N molar ratio of 200:1 for R. arrhizus 2582. When nitrogen limitation is not desired, phosphorus limitation can be used instead (Riscaldati et al. 2000). Trace metal concentrations of 500 ppm, 4 ppm, and 100 ppb for Mg++, Zn++, and Fe++, respectively, were found optimal for the formation of small (1 mm) spherical pellets that produced high concentrations of fumaric acid (36 g L−1; Zhou 1999).
Because CO2 is needed for the oxaloacetate formation from pyruvate by pyruvate carboxylase, the addition of calcium carbonate (CaCO3), which is used in many cases as a neutralizing agent, seems also important as a CO2 source during the production phase of the fermentation. However, in case that no CO2 or carbonate is added, the complete catabolism of a mole of glucose via the citrate cycle provides 6 mol of CO2 that may be used for pyruvate carboxylation (see Fig. 3). In this case, the maximum theoretical yield would be 1.5 mol of fumaric acid per mole of glucose. This theoretical value is close to the value of 1.32 mol of fumaric acid per mole of glucose reported by Cao et al. (1996), who optimized a fermentation process without CO2 or carbonate feeding.
Neutralizing agents
Continuous neutralization of the pH has been necessary to obtain optimal yields in fumaric acid production by fermentation. Preferably, CaCO3 has been used as a neutralizing agent, but at the same time, this compound is causing viscosity problems due to the low aqueous solubility of calcium fumarate [21 g/l at 30°C (Gangl et al. 1990)] that precipitates as a fermentation product. Furthermore, the cells can interact with the precipitated product, as has been found for R. oryzae, resulting in a highly viscous suspension. This has a detrimental effect on the rate of oxygen transfer which can be achieved, and hence, the fermentation might fail due to oxygen-limitation problems. Therefore, replacement of CaCO3 by other neutralizing agents like Na2CO3, NaHCO3, Ca(OH)2, and (NH4)2CO3 has been studied by different authors (Gangl et al. 1990; Riscaldati et al. 2000; Zhou et al. 2002). However, these studies showed that the fumarate production rates are the highest when CaCO3 is used as a neutralizing agent.
Because of the high solubility of sodium fumarate, fermentative production of fumarate using Na2CO3 as neutralizing agent leads to cheaper downstream processing than when CaCO3 is used. This is due to the fact that the fermentation product, sodium fumarate, has a higher solubility than CaCO3, and hence, there is no need of heating to recover the fermentation product (see Fig. 4). In addition, cells can be reused (Gangl et al. 1990; Zhou et al. 2002). A similar situation was obtained when Rhizopus growth was limited by phosphorus so that (NH4)2CO3 could be used as neutralizing agent (Riscaldati et al. 2000). Nevertheless, it has been argued that a fermentation process without the use of neutralizing agents, and at the same time preventing product inhibition, will improve the economics of the general process (Gangl et al. 1990). However, when a high-yield process is developed without a carbonate as neutralizing agent, the required CO2 must be supplied by other sources. Fumaric acid fermentation systems without the use of neutralizing agents have been studied in the past and will be discussed in the following sections.
Fig. 4Flow-sheet for fumaric acid production via fermentation. a Formulation tanks containing glucose and nutrients; b Seed fermentor; c Production fermentor; d Filter; e Acidification tank; f Filter; g Rotary dryer (adapted from Gangl et al. 1990)
With respect to fumarate salts inhibition, Rhodes et al. (1962) reported that production of soluble sodium or potassium fumarates was inhibited when the concentration of fumarate reached values of 34–40 g L−1 using R. arrhizus as a producer strain. Nonetheless, Gangl et al. (1990) found that addition of sodium fumarate (71.3 g L−1) was not inhibiting the same strain, although the cells needed 35 h to adapt to the high sodium fumarate concentrations. On the other hand, the free fumaric acid does inhibit its own production because the accumulated protons in the production medium decrease the pH, thus exerting a progressive inhibitory effect on fumaric acid production (Riscaldati et al. 2000). At low pH, excreted fumaric acid will passively diffuse back through the plasma membrane of the fungus decreasing its intracellular pH, and due to this phenomenon, the fermentation fails. Proposed methods to enable carboxylic acid fermentation at low pH are: genetic engineering of acid tolerant organisms such as yeasts and in situ product removal techniques (Cao et al. 1996; Schügerl 2000; van Maris et al. 2004).
Morphology and oxygen-transfer problems
One of the difficulties of fermenting Rhizopus species is the morphology of these fungi. Rhizopus species tend to grow on the walls and on the stirrer of the reactor, and sometimes, clumps are formed. Therefore, the fermentation can suffer from oxygen limitation in particular when calcium fumarate is present. One way to solve this problem is to control the growth of the fungi and their morphology.
A way to minimize oxygen mass transfer limitation to the cells is to stimulate formation of small spherical cell pellets (Zhou 1999). Small pellets can reduce clump formation during fermentation, and even if CaCO3 is present, the viscosity of the broth can be reduced. Moreover, pellets can facilitate the performance of a biomass retention system. For R. oryzae, low initial pH values for the cultivation media favored pellet formation, and good fumaric acid yields have been reached (Zhou et al. 2000).
In another morphology improvement study, Cao et al. (1997) used a rotary biofilm contactor (RBC) as fermentor with immobilized R. oryzae to produce fumaric acid. CaCO3 was used as neutralizing agent during this experiment. During the fermentation, the discs with immobilized cells were rotating, moving the cells from the gas phase of the fermentor to the liquid phase and back again (Cao et al. 1997). When the cells are exposed to the air, high oxygen transfer rates can be reached, while the cells can take up substrate and excrete the produced fumaric acid when they are submerged. In this system, additional agitation was not needed. The volumetric productivity was very high, compared to the volumetric productivity of an equivalent stirred vessel fermentor setup (see Table 1). A disadvantage of this system could be the scalability potential of the RBC fermentor.
Immobilization techniques for Rhizopus species have been investigated to open the possibility of a continuous operation mode for fumaric acid production and to reduce oxygen transfer problems as well. Buzzini et al. (1995) searched for the most suitable support for cell immobilization in fluidized-bed reactors during fumaric acid fermentation. Cork, expanded polystyrene, expanded clay, and wood shavings were investigated. With 6 mm pieces of cork, the highest titer, 24.1 g fumaric acid l−1 in 144 h, was achieved, which is comparable to the titer of 37.7 g l−1 after 166 h under conventional submerged conditions (Buzzini et al. 1995). The performance of a semi-continuous process was investigated with R. arrhizus immobilized on 5 mm cubic particles made of polyurethane sponge (Petruccioli et al. 1996). These particles were used in repeated batch fermentations in a fluidized-bed reactor (48 h, eight times). In the optimized process, the fumaric acid titer was 12.3 g l−1, and the volumetric productivity was 0.256 g l−1 h−1.
Furthermore, as the oxygen mass transfer resistance through the boundary layer on the liquid side of the gas–liquid interface can affect the interfacial oxygen transfer from the gas phase to the liquid phase, pressure pulsation was applied in a stirred tank fermentor using R. oryzae (Zhou 1999) to reduce this resistance. Mass yield and volumetric productivity of fumarate were 70.1% and 0.99 g L−1 h −1, respectively, which were higher than for traditional stirred tank fermentations. The same organism was studied in an airlift loop reactor as a fermentation system for fumaric acid production (Du et al. 1997). Here, the airlift loop reactor with porous sparger produced favorable conditions for mass transfer, and also, higher yields and productivities were reached than in stirred tank fermentations.
Integrated fermentation and recovery of fumaric acid
Figure 4 presents a flow scheme proposed for fumaric acid production by batch fermentation (Gangl et al. 1990). Glucose and mineral salts are fed to the fermentor. The nitrogen source solution, (NH4)2SO4, is sterilized separately and fed to the seed fermentor. The harvested broth containing sodium fumarate (cells and trace amounts of Na2CO3) is filtered to remove the cells and then acidified by H2SO4 to pH 1. After acidification, fumaric acid precipitates out of the solution and is sent to a rotary dryer to be completely recovered (Gangl et al. 1990). When CaCO3 is used as a neutralizing agent instead of Na2CO3, additional heating after the fermentation was supposed to be required to dissolve calcium fumarate and the excess of CaCO3 that usually stick to the cells. This problem leads to a tedious and expensive downstream processing.
In the downstream processing field, recovery techniques have not so much been studied for fumaric acid producers in submerged cultivations as it has been done for related fermentation products like succinic acid (Zeikus et al. 1999), citric acid (Heinzle et al. 2006), and lactic acid (Joglekar et al. 2006). Recovery systems like reactive extraction and membrane electrodialysis have not been studied yet. However, simultaneous fermentation and adsorption have been studied to remove fumaric acid during its formation to control fermentation pH at desired values and avoid product inhibition (Cao et al. 1996; Zhou 1999).
Cao et al. (1996) used a rotary biofilm contactor (RBC) setup as fermentor for R. oryzae, in combination with an adsorption column. The produced fumaric acid was removed from the broth by the adsorption column in a recycle loop, reducing product inhibition and thus increasing the production rate and sustaining cell viability. Polyvinyl pyridine (PVP) anion exchange resin in the hydroxide form was selected as adsorbent because it yielded the highest loading capacity for fumaric acid (0.31 g g−1 dry wt). The RBC, coupled with the adsorption column, increased the fumaric acid productivity significantly to 4.25 g l−1 h−1 because the total fermentation time was much less than in traditional stirred tank fermentations (see Table 1). This volumetric productivity and the yield are the highest reported in literature. In this integrated system, the constant removal of the fumarate produced and the liberation of OH− from the adsorption column kept the fermentation pH at 4.5. The fumarate was desorbed from the adsorption resin by using 0.4 M NaOH so additional processing will be necessary to convert the sodium fumarate into neutral fumaric acid. Addition of extra CO2 was not necessary due to the fact that during the production stage, the biofilm was exposed to sterile air that was present in the headspace of the RBC enhancing the opportunity for CO2 fixation by the biofilm.
A novel product recovery process by an ion exchanger resin was developed by Zhou (1999). Fumarate was recovered from the fermentation broth without the use of neutralizing agents, while keeping the pH at 5, by cycling the broth over a column of a resin (Amberlite IRA-900 with OH- as counterion). After eluting the loaded column with ammonium hydroxide, ammonium fumarate solution was obtained. This was passed through a Y-zeolite column that retained the ammonium and liberated the fumaric acid. The ammonium hydroxide solution can be recovered and recycled by thermal regeneration of the zeolite (Zhou 1999). However, this integrated process did not surpass yield values obtained in a stirred tank fermentor when fumaric acid fermentation was controlled by CaCO3 under pressure pulsation (see previous section), but a higher productivity value of 1.09 g L−1 h−1 was reached (Zhou et al. 2000).
Economics of fumaric acid production
In a theoretical study, Gangl et al. (1990) compared a fermentation and a benzene-based petrochemical process with respect to economy. The fermentation process taken for this has been described in the beginning of the previous section. Although Na2CO3 was assumed as a neutralizing agent in this study, the productivity (2 g l−1 h−1) and yield (0.82 g g−1) of the fumaric acid fermentation with CaCO3 obtained in a previous study were used (Ng et al. 1986).
This economic evaluation showed that the fermentation route was less favorable than the petrochemical route (economic evaluation included upstream and downstream sections). In particular, the raw material costs were higher for the fermentation process than for the petrochemical route. It was concluded that the fermentation route could become competitive with the petrochemical route if oil prices were around 61 $/barrel. The latter price has been reached in the recent years (http://tonto.eia.doe.gov/dnav/pet/xls/PET_PRI_WCO_K_W.xls), while the productivity of the fermentation process has been improved significantly as is shown in Table 1. Besides, one can roughly calculate from Gangl’s study that the assumed sugar price was around 0.6 $/kg of glucose, which is high compared to the current price (see Table 2). Therefore, if the current glucose price is assumed in the calculations of Gangl’s study, one can expect lower raw material costs for the fermentation route. On the other hand, the petrochemical route has also been improved significantly, now using butane instead of benzene. This will be reflected in the current maleic anhydride price, which is shown in Table 2.
Table 2Comparison between petrochemical and fermentation route for fumaric acid productionParameterPetrochemical routeFermentation routeRaw materialMaleic anhydrideGlucoseReaction temperature (°C)90–10035Raw material price ($/kg)1.46–1.63 (Anonymous 2007)0.46aProduct yield (% w/w)112 (Lohbeck et al. 1990)85 (Cao et al. 1996)ahttp://www.ers.usda.gov/briefing/sugar/data.htm; for 4th quarter of 2006
The simple comparison of petrochemical and fermentation routes for fumaric acid production given in Table 2 suggests that the lower raw material price of the fermentative production might compensate the higher yields of the petrochemical production from maleic anhydride, and fermentation may become an economically viable alternative.
Conclusions and future prospects
Due to increasing prices of fossil feedstock, fermentatively produced fumaric acid could become a cheaper alternative to the petrochemically based maleic acid as unsaturated dibasic acid in polyester resins in the nearby future. Based on the available literature, the microorganism with the highest productivity and yield of fumaric acid appears to be R. oryzae. This organism produces fumaric acid via a combination of the citrate cycle and reductive pyruvate carboxylation. In addition, the fumaric acid production by fermentation can be improved if the use of neutralizing agents is reduced or avoided and the morphology of the fungi is optimized. However, avoiding the use of a neutralizing agent will lead to product inhibition; therefore, it would be useful if metabolic engineering is applied to achieve fumaric acid production in suspended, acid-resistant microorganisms such as yeast. Another way to solve product inhibition problems is applying in situ removal of fumaric acid during the fermentation.

fumaric acid
fermentation
rhizopus species
product recovery
maleate isomerase


Topiramate in the prevention of pediatric migraine: literature review

Pediatric migraine is a disabling condition, which can cause a significant impact on quality of life. Currently, no drugs have been approved by the FDA for its preventive treatment. Our aim was to review the medical literature concerning the efficacy and tolerability of topiramate in the prophylactic treatment of migraine in children and adolescents. A total of five papers were reviewed: two randomized controlled trials (RCTs), a post-hoc subset analysis of adolescents who had been included in three RCTs carried out on adults and two open studies. Topiramate has been proven to reduce headache frequency and the accompanying disability. The frequency of side effects varied considerably among studies, the most frequent being weight loss, anorexia, abdominal pain, difficulties in concentrating, sedation and paresthesia. Since these adverse events, although often transitory, may be distressing for the child, we strongly recommend to assess the disability caused by the migraine episodes before deciding to initiate a prophylactic treatment. Nevertheless, dropout rates due to side effects in the studies were very low.
Introduction
Pediatric migraine is a disabling condition, which can cause a significant impact on quality of life. It can negatively influence the child’s school performances, social and family life and often causes school absenteeism. Migraine prevalence in childhood ranges from 2.7 to 10% [1]. About 3–5% of school-aged children suffer from migraine and this proportion gradually increases to 20% through adolescence. Initially, there is a slight male predominance; however, through adolescence, there is a shift toward female predominance, which remains through adulthood [2, 3].
Management of pediatric migraine includes lifestyle changes (in order to avoid foods, habits or enviromental factors that may trigger a migraine attack), the use of abortive medications and preventive measures, which can be either nonpharmacological or pharmacological. Guidelines addressing the indications for the use of preventive measures in children are lacking, but to date, most authors recommend preventive measures in children when headache frequency exceeds three to four episodes per month and/or the attacks are significantly disabling, as measured in scoring systems such as the Pediatric Migraine Disability Assessment Scale (PedMIDAS) [4, 5]. PedMIDAS is a validated six-item questionnaire based on the adult MIDAS tool [6] with developmentally appropriate changes and adjustments for childhood lifestyle. The questions deal with the impact of headache on school (school day absences, partial day absences, functioning at 50% or less ability in school), on household acitivities (ability to perform homework and chores) and on social functioning (including sports) [7, 8]. The assessment of migraine disability is particularly important in children to correctly evaluate and treat migraine.
Currently, no drugs have been approved by the Food and Drug Administration (FDA) for the preventive treatment of pediatric migraine. Topiramate is an antiepileptic drug, which has been approved by the FDA in the United States and in many other countries for the preventive treatment of migraine in adults and for the treatment of partial-onset seizures and primary generalized tonic–clonic seizures as add-on therapy in children as young as 2 years. Our aim was to review the medical literature concerning the efficacy and tolerability of topiramate in the prophylactic treatment of migraine in children and adolescents.
Materials and methods
We searched Pubmed (1966–July 2007) using the following keywords: “topiramate” + “headache” or “migraine” + “children” or “adolescents” or “pediatric.” We included randomized controlled trials (RCT), open-label trials and retrospective studies. We excluded case-reports and studies conducted on less than 10 patients. We classified the studies in accordance with the AAN classification of evidence for therapeutic intervention (Table 1) [9]. A total of five papers were reviewed.
Table 1AAN classification of evidence for therapeutic interventionClass I: Prospective, randomized, controlled clinical trial with masked outcome assessment, in a representative population.The following are required: (a) Primary outcome(s) is/are clearly defined (b) Exclusion/inclusion criteria are clearly defined (c) Adequate accounting for drop-outs and crossovers with numbers sufficiently low to have minimal potential for bias (d) Relevant baseline characteristics are presented and substantially equivalent among treatment groups or there is appropriate statistical adjustment for differencesClass II: Prospective matched group cohort study in a representative population with masked outcome assessment that meets (a)–(d) above OR a RCT in a representative population that lacks one criteria (a)–(d).Class III: All other controlled trials (including well-defined natural history controls or patients serving as own controls) in a representative population, where outcome is independently assessed, or independently derived by objective outcome measurement.Class IV: Evidence from uncontrolled studies, case series, case reports, or expert opinion.
Results
We found two RCTs, a post-hoc subset analysis of 51 adolescents (12–17 years) who had been enrolled in three pivotal RCTs of topiramate for migraine prophylaxis in adults and two open studies.
In a recent Class I randomized, double-blind, placebo-controlled trial [10], 44 children were randomized (in a 1:1 ratio) to receive either placebo or topiramate (titrated to 100 mg a day) for 12 weeks. Participants had a diagnosis of migraine without aura (according to 2004 International Headache Society criteria) [11] and a frequency of two or more headaches per month for 3 months before entering the study. Children with comorbid medical associations or who were already on migraine prophylaxis were excluded. Primary outcome measures were the reduction in migraine frequency and severity; secondary outcome measures included number of analgesics taken and the functional disability. Migraine monthly frequency decreased from 16.14 (±9.35) at baseline to 4.27 (±1.95) at the end of the study in the treated group as compared with a decrease from 13.38 (±7.78) to 7.48 (±5.94) in the placebo group (P = 0.025). The percentage of topiramate-treated subjects showing >50% reduction in monthly migraine days during the double-blind phase was 95.2% as opposed to 52.4% in the placebo group (P = 0.002). The Pediatric Migraine Disability Assessment Score (PedMIDAS) also decreased significantly (P = 0.003) as did school absenteeism (P = 0.002). Side effects were rated mild to moderate, did not interfere with daily activities or cause any dropouts and included weight-loss (mean loss of 0.3 kg) in 81% of patients, paresthesia (23.8%), loss of appetite (23.8%), lack of concentration (19%), sedation (19%) and abdominal pain (14.3%). This seems a well-performed trial in which the mean monthly migraine frequency reduction is clearly evident in the topiramate-treated group. Patients treated with topiramate suffered at least five episodes a month before entering the study (range 5–30); a preventive treatment was, thus, clearly indicated. The reduction in the PedMIDAS score indicates that the drug side effects (difficulties in concentrating, sedation) interfered with school, household or social activities to a lesser extent than the migraine episodes.
The other Class I, randomized, double-blind, placebo-controlled trial [12] was carried out on 162 children (age 6–15 years) who were randomized in a 2:1 ratio to receive either topiramate (which was titrated over 8 weeks to 2–3 mg/kg per day and maintained for 12 weeks) or placebo. Eligible criteria were the children with migraine with or without aura, weighing more than 20 kg, who experienced 3–10 migraine days/month for the 3 months prior to the screening and during the 4-week prospective baseline phase. Exclusion criteria included chronic migraine, analgesic overuse and previous failure of ≥2 adequately dosed migraine preventive medications. One hundred and fifty-seven subjects were included in the intention to treat (ITT) population, defined as randomized subjects who had received at least one dose of study medication and had at least one postbaseline efficacy assessment, and a total of 131 children completed the study (per-protocol population); of these, 108 were treated with topiramate. Primary outcome measure was the reduction of migraine days per month in each treatment group of the ITT population. During the double-blind-phase, relative to the 4-week prospective baseline phase, there was a reduction of 2.6 ± 2.6 migraine days per month in the topiramate group compared with a mean reduction of 2.0 ± 3.1 migraine days per month in the placebo group (P = 0.061). In the per-protocol population, the reduction in monthly migraine days (2.8 ± 2.4 as opposed to 2.2 ± 2.1 in the placebo group) reached statistical significance (P = 0.033). During the last 28 days of treatment, the mean number of monthly migraine was reduced by 3.1 ± 2.6, as opposed to 2.4 ± 2.8 in the placebo group (P = 0.023). In the treated group, 32% of patients experienced a ≥75% reduction in mean monthly migraine days compared with a 14% reduction in the placebo group (P = 0.02). The dropout rate for adverse events was 6.3% for the topiramate group and 4% for the placebo group. In the treated group, the most common adverse events were anorexia (13%); weight decrease (10.2%), with a mean loss of 1.4 ± 2.6 kg (as opposed to 0.7 ± 3.9 kg in the placebo group); abdominal pain (10.2%); paresthesia (8.3%); and somnolence (8.3%). Serious adverse events occurred in four topiramate-treated patients and included infection (n = 2), severe migraine (n = 1) and suicidal ideation (n = 1).
In this study, the mean monthly migraine frequency reduction seems less evident when compared to the previous study [10], probably because migraine frequency at baseline was lower (range 2–9 in the ITT topiramate population) and children with chronic migraine were excluded. An important aspect is that the reduction is more evident in the last 28 days of treatment, indicating that it may take a few weeks before treatment with topiramate reaches its maximum efficacy.
Winner et al. [13] performed a post-hoc subset analysis on 51 adolescents (12–17 years) who had been enrolled in three pivotal trials of topiramate for migraine prophylaxis in adults. Patients were to have had between 3 and 12 migraine attacks, and no more than 14 headache days per 28 days during the 3 months prior to the screening and during the 4-week prospective baseline phase. Also excluded were patients who overused analgesics or who had failed to respond to two or more adequate prophylactic treatments. Topiramate was administered at the dosages of 50, 100 or 200 mg a day for 26 weeks. When compared to baseline, there was a mean monthly migraine frequency reduction of 46, 63 and 65%, respectively, as opposed to a mean 16% reduction in the placebo group (P = 0.07, P = 0.02, P = 0.04, respectively). The most common adverse events (in the group treated with 100 mg a day) were as follows: paresthesia (38%), upper respiratory tract infections (23%), weight decrease (15%), abdominal pain (15%), anorexia (8%) and somnolence (8%). There were no dropouts in the group treated with topiramate. The incidence of adverse events was higher in the group treated with topiramate at the dosage of 200 mg, which did not appear to confer additional efficacy as opposed to the dosage of 100 mg a day.
One class IV study [14], assessing the efficacy of topiramate for pediatric migraine, included 97 children, 75 of which were reevaluated at a first follow-up visit (after 88.7 ± 35.7 days) and 41 at a second follow-up visit (after 203.1 ± 45.6 days). Included were children reporting more than three headaches per month. Topiramate was administered at the dose of 1.4 ± 0.74 mg/kg/day and headache frequency decreased from 16.5 ± 10 headaches/month to 11.6 ± 10.2 headaches/month (P < 0.001) at the first follow-up visit, with 43.1% of the patients experiencing a 50% or greater reduction in the number of headaches. By the second follow-up visit, headache frequency had decreased to 9.4 ± 8.4, with 56.1% of the patients experiencing a 50% or greater reduction in the number of headaches. Mean headache severity, duration and accompanying disability were also reduced. Disability was measured using the PedMIDAS score: a 50% reduction occurred in 48.6% of the patients at the first follow-up visit and in 62.5% of patients at the second follow-up visit. One quarter of patients complained of side effects, which declined in subsequent visits and included cognitive changes (12.5%), weight loss (5.6%) and sensory symptoms (2.8%). A possible bias of this study is that 50.7% of patients were on additional prophylactic medication (most often amitriptyline or divalproex).
In an open, prospective case series (class IV) study [15], topiramate was administered to 24 children with migraine, who had failed to respond to other prophylactics, at the mean dosage of 3.5 ± 1.7 mg/kg for 4 months. Authors report a reduction in the duration and in the intensity of headaches; headache frequency reduction, however, did not reach statistical significance. Adverse events (emotional instability, paresthesia, anorexia, asthenia, weight loss) were experienced by one-third of patients. In our opinion, the baseline headache frequency (3.6 ± 2.7 a month with a range of 1–12) and duration (2–6 h) did not necessarily constitute an indication for prophylactic treatment in all treated cases.
Unfortunately, the studies we reviewed were not homogeneous with regard to inclusion and exclusion criteria and with regard to outcome measures such as the “responder rate,” which was not considered in all studies. Furthermore, the degree of disability caused by the migraine episodes, which is essential in the decision to initiate prophylactic treatment, was not assessed in all studies.
Discussion
Management of pediatric migraine can be tricky, seeing as no pharmacological treatment is formally approved or indicated in the prophylactic treatment of migraine in children and adolescents. A recent review of the pharmacological treatment options in pediatric migraine [16] found only flunarizine, which is not available in many countries, to be effective in rigorous controlled trials. Authors also concluded that there is conflicting evidence regarding propanolol and trazodone, while data from uncontrolled studies suggest cyproheptadine, amitriptyline, naproxen and antiepileptic drugs (topiramate, valproic acid and gabapentin) to be effective. Since this review was published, two randomized, placebo-controlled trials evaluating topiramate in the preventive treatment of pediatric migraine have been carried out [10, 12].
Taken together, data from the papers we reviewed suggest that topiramate is effective in the preventive treatment of pediatric migraine. Furthermore, topiramate dosages, which seem to be effective in the treatment of migraine in children and adolescents (2–3 mg/kg/day) are much lower than those indicated for the adjunctive treatment of epilepsy (5–9 mg/kg/day) in children as young as 2 years.
As regards its safety and tolerability, serious adverse effects were very rare (one patient with suicidal ideation), though the risk of depression must be kept in mind. The percentage of patients experiencing side effects varied greatly among the studies, the most common ones being weight loss (which, in the RCTs [10, 12], was present in 81 and 10.2% of patients, respectively), anorexia (23.8 and 13%, respectively), abdominal pain (14.3 and 10,2%), difficulties in concentrating, somnolence/sedation (19 and 8.3%) and paresthesia (23.8 and 8,3%). Side effects tended to decline over time and dropout rates due to side effects were very low (range 0–6.3%). Since these adverse events, although often transitory, may be clinically significant and distressing both for the child and its family, the decision to initiate treatment must be carefully weighed and should take the degree of disability caused by the migraine episodes into account.
An important aspect, which emerged from some of the studies, is a significant decrease in school absenteeism due to migraine [10] and a reduction in the disability caused by migraine (as assessed by PedMIDAS), leading to an improvement in the childrens’ quality of life [10, 14].
In conclusion, topiramate seems to be a promising therapeutic option, though, clearly, further controlled trials are needed to confirm this data, as are studies comparing different drugs.

topiramate pediatric migraine children headache


Characteristics of dynamic processing in the visual field of patients with age-related maculopathy

Purpose To investigate the characteristics of dynamic processing in the visual field of patients with age-related maculopathy (ARM) by measuring motion sensitivity, double-pulse resolution (DPR), and critical flicker fusion.
Introduction
Age-related maculopathy (ARM) is the most common cause of loss of central vision in industrialized countries [17, 26, 30, 33, 34, 62]. Almost all clinical assessments of the consequences of ARM for basic visual functions and quality of life refer to foveal or macular vision. However, a few studies have shown that retinal defects can occur beyond 10° eccentricity, which can be considered the maximum radial extent of the macula. Sunness et al., for instance, described histopathological anomalies outside the central retina, whereas sensitivity was unchanged in traditional static perimetry [55].
Curcio et al. described that, beyond general age-related photoreceptor loss, five out of six ARM-affected donor eyes showed cone and rod loss in parafoveal regions [12]. The latter can be expected to reduce dark adaptation performance, which was confirmed up to 25° eccentricity by Brown et al. [9]. In addition, ARM affects temporal aspects of visual information processing. Mayer et al. found reduced temporal resolution of ARM patients as measured by foveal flicker sensitivity with a stimulus of 2.8° diameter [40, 41]. Brown and Lovie-Kitchin showed reduced flicker resolution in the fovea and at 10° and 20° eccentricity and concluded that “the functional effects of ARM are not confined to the central retina, but affect a large region of the visual field” [11]. Falsini et al. found cone-mediated flicker sensitivity (CFS) losses in ARM by evaluating the focal electroretinogram (FERG) as a function of flicker modulation depth in the macula (9° radius) [13].
In summary, the results of these studies show age-related as well as ARM-related functional loss of dynamic processing. However, whether the loss is confined to the macula has not been definitively answered. A further question is whether a loss outside the macula is specific to temporal resolution or whether other temporal aspects of retinal processing, such as motion sensitivity, can also be affected beyond the macula in patients suffering from ARM. Furthermore, we asked whether the impairment is confined to the magnocellular system or also involves the parvocellular pathway.
Methods
Motion contrast threshold
Experimental setup
The experimental setup allowed measurements of peripheral motion perception up to 60° eccentricity under photopic conditions (luminance = 75 cd/m2). It consisted of a white, semicircular (180°) plastic screen (radius 90 cm, height 60 cm) with a rectangular opening in the center. A flat-screen LCD display was mounted behind the opening for the presentation of the test patterns. The subjects sat in front of the screen at a distance of 90 cm and monocularly fixated one of the fixation marks in primary view (fixation crosses with diameter = 4 cm and bar width = 0.5 cm). The marks were attached to the plastic background at 10°, 20°, 30°, 40°, and 60° eccentricity (Fig. 1). Fixation was monitored by a mirror mounted below the LCD display. Thus, the examiner could monitor the subject’s eye movements during the presentation of a stimulus. Additionally, the mirror could be moved horizontally to allow a clear view of the patient’s eye, even in extremely peripheral positions. We are confident that the examiner was able to detect saccadic eye movements of at least 5° amplitude, which is still only half of the distance between two measuring locations.
Fig. 1Experimental set up (top view). The subject’s chair was rotated to maintain primary gaze for all tested fixation marks. a LCD monitor screen. b Semicircular plastic screen. c Fixation crosses (diameter = 4 cm, bar width = 0.5 cm). d Chin rest for stabilizing the head and keeping the viewing distance constant. e Subject’s right or left eye
Monitor calibration
To achieve specified stimulus luminance and contrast, the monitor’s gamma curve (screen luminance vs. gray value) needs to be taken into account during stimulus presentation [4, 51, 52, 54]. We measured this for our monitor using Irtel’s PXL software to set the screen uniformly at the 256 possible gray levels and measured center screen luminance with a digital luminance meter (Gossen Mavomonitor G04068, Nürnberg, Germany) [25]. A second order function was fit to the luminance data by non-linear regression (r2 = 99%):
where L is center screen luminance in cd/m2 and g is the uniform gray value (range 0–255). These function parameters are incorporated in our stimulus presentation software (see below). Target contrast is specified in the Michelson measure, .
Design of the motion stimulus
The test software for motion perception was custom-developed for these experiments and consists of two separate modules, one for stimulus generation and another for running the actual test program. Since threshold measurements are much faster and more reliable using four-alternative forced choice than two-alternative forced choice tasks, we employed four directions of motion (up, down, left, and right) to be discriminated by the subject. The stimuli were plaids composed of two orthogonal Gabor patterns of 45° left and right tilt [15]:
where L and L0 are the luminances of pattern and background, respectively,  is the square of radial diameter of the Gaussian envelope (the value where the amplitude has decreased to 1/e), and ϕ(t) is the spatial phase at time t (i.e. the shift within the envelope). For the plaids used here, we chose σ = 0.8, which results in about 1.5 visible cycles (Fig. 2).
Fig. 2Gabor stimulus with double sinusoidal modulation in spatial quadrature. The entire patch was stationary, while the plaid pattern moved within the envelope in one of four possible directions. In addition, the stimulus was temporally modulated by a Gaussian envelope function, so that it slowly appeared out of the medium grey background and then merged back into it
The subject’s task was to identify the direction of motion. Watson found that thresholds of motion detection and motion discrimination in healthy subjects are equal at sufficiently low spatial frequencies (e.g. 2 cpd) for both slow (1.5 Hz) and fast (12.4 Hz) movement of the test pattern [63]. Therefore, we used a low spatial frequency of 0.65 cpd and an intermediate speed of 5.71 °/s (corresponding to 3.75 Hz local luminance change) for the motion stimulus. The size of the test pattern at the viewing distance of 90 cm was 3.8°. To minimize attracting transient attention by abrupt pattern onset, the test pattern gradually appeared and disappeared by using a Gaussian temporal envelope [27, 36, 37]. Due to the small changes between frames in these stimuli, “tearing” of the image was extremely unlikely and was indeed never observed [16].
Adaptive algorithm
Monocular contrast thresholds for motion perception were measured along the horizontal meridian in the nasal and temporal visual field at 10°, 20°, 30° and 40° eccentricity and in the temporal visual field additionally at 60°. The initial contrast value for all eccentricities was 30% (1.48 log %-contrast). For each eccentricity, the threshold was determined twice. Two blocks of nine measurements each were taken and the arithmetic mean calculated from the two results. The adaptive algorithm for threshold determination was based on Kesten and was modified according to Kaernbach to allow the additional response alternative “I don’t know” [28, 29, 32, 58]. This paradigm, termed “unforced choice task” by Kaernbach, has been shown to be beneficial, especially for subjects who are unfamiliar with psychophysical testing procedures.
Kesten’s algorithm is of the staircase kind, i.e. the intensity of the next presentation depends on the previous response only, not on the entire history or a longer sequence of preceding responses [32]. After a correct response, the log contrast of the stimulus was decreased by one incremental step, and after an incorrect response, it was increased by 1.67 incremental steps (5/3). It thereby converged on the 62.5%-correct point (5/8), i.e. the point of inflection ϕ on the psychometric function (of log contrast) in a four-alternative forced-choice task. The ratio V=5/3=1:1.667 of up-down intensity changes results from Kesten’s algorithm given the number n of response alternatives (four) by
where ϕ is the probability at threshold or point of convergence,
with guessing rate γ  = 1/n = 1/4. (c.f. Treutwein’s equation 16, the last term (Zn–ϕ) [58]). When the subject gave the indecisive response “I don’t know”, the intensity of the next stimulus was increased by one incremental step, as specified by Kaernbach’s extension which requires the specification of V as above [28]. The step width in Kesten’s algorithm is reduced by a factor of m/(m + 2) after a change in response category (correct-to-incorrect or vice versa) where m is the number of reversals (Treutwein’s eq. 16). Since Kaernbach’s extension requires the above-mentioned fixed ratio of up-down intensity changes, we used a step width reduction only every other reversal (i.e. m even). The Kesten rule is applied to log contrast, i.e. log Cn + 1= (logCn)±sn (where sn is the step width), which is equivalent to contrast being multiplied or divided by the antilog of step width.
Double-pulse resolution
Treutwein and Rentschler developed a hardware and software combination for the measurement of double-pulse resolution (DPR) which allows simultaneous measurement at multiple visual field positions [60]. The technique has been used in experiments on patients with multiple sclerosis and glaucoma, and in an extensive study characterizing the temporal properties of the visual field across the life span [46, 48–50]. The experimental setup allowed (limited by screen size) measurement of DPR up to 20° eccentricity, i.e. not as far into the periphery as for the motion contrast thresholds but well beyond the macular region.
Test setup
An analogue 15″ x-y-z display was used for stimulus presentation (Hewlett Packard model 1310, i.e. a CRT-display without a raster-scan generator), driven by a temporary buffer that stores the point coordinates and generates the control voltages for the display (“point plot buffer”; G. Finlay, Edmonton, Canada). Temporal control could be extended into the low microsecond range, so that the temporal resolution was better by a factor of 1,000 than in conventional setups [60]. A computer (PC) was used for running the experimental software that controlled stimulus generation, adaptive procedure, and data acquisition. The experimenter recorded the subject’s responses via the computer keyboard.
Stimulus characteristics
The tests were performed monocularly at 30 cm viewing distance with a diagonal screen diameter of 40 cm. The stimuli in Treutwein’s technique are eight squares of 1.15° × 1.15° visual angle (5 × 5 pixels) each, arranged in circles at 5°, 10° , and 20° eccentricity around a ninth square in the center [60]. Figure 3 shows the stimulus time course. Eight of nine squares are continuously present for 80 + γ + 280 ms. The target, one randomly selected stimulus, is switched off after 80 ms and then on again after a variable time gap γ. The subject’s task was to detect the gap and indicate the target position on the screen, which makes it a nine-alternative forced choice task. In supra-threshold trials, its location can be recognized as a short flicker of the target. A central fixation cross was presented continuously between stimulus presentations and was switched off 50 ms before the beginning of a new trial. The squares’ luminance was 215 cd/m2; subjects were light-adapted and the display background luminance was held constant in the low photopic range (luminance ∼15 cd/m2).
Fig. 3Double-pulse stimulus characteristics (figure modified after Poggel and Strasburger, originally from Treutwein and Rentschler [46, 60]. a, b Time line diagram of stimulus display. c Stimulus positions (5°, 10°, and 20° eccentricity)
Gap duration was varied by an adaptive thresholding algorithm (“YAAP”), based on maximum-likelihood psychometric function fitting [20, 59].
Flicker frequency analyzer
For measurement of foveal flicker fusion frequencies we used the “Wiener Testsystem”, a commercial computer-based diagnostic system with an emphasis on assessing driving fitness (Schuhfried, Moedling/Vienna, Austria). The system was chosen as a reference, because it is an established standard system in Germany used for psychological testing; due to its construction it allows foveal measurement only. Measurements were performed by presenting a circular flickering red light stimulus (1.2° diameter; luminance = 270 cd/m2; wave length 655 nm) on a white background in a tubular viewer. Thresholds were determined by the method of limits, i.e. the frequency of the flickering light is increased until permanent light is perceived and is then decreased until the light is once again perceived as flickering.
The subject indicated the perceived changes by a keystroke whereupon the critical frequency was recorded. The separate arithmetic means of the critical frequencies determined in the ascending and descending series are referred to as fusion frequency and flicker frequency, respectively. Each test cycle consisted of five training cycles, immediately followed by eight measurement cycles. For further analysis, the mean of the fusion frequency and the flicker frequency was calculated for each subject, which is referred to as the critical flicker fusion frequency (CFF).
Color perception
The Lanthony D-15 color vision test (desaturated) was used for the examination of color perception. The test consists of 15 colored chips, which are used to determine the subject’s capability to discriminate hues. At 50 cm viewing distance, every chip has a diameter of about 1.5°. The subject sorts the arbitrarily shuffled chips according to the arrangement in the color cycle. The chosen sequence was then documented in a test protocol and the color confusion score (CCS) and crossings over the color space were determined [1]. The measurement was done once under constant lighting conditions (color temperature ∼4000 K).
Subjects
We recruited three groups of subjects for this study: 14 subjects with ARM (19 eyes, mean visual acuity: 0.29, SE ± 0.048; ∼20/63), a control group of age-matched subjects with healthy eyes (14 subjects; 18 eyes, mean acuity: 0.65, SE ± 0.057; ∼20/32), and another control group of 7 young subjects (8 eyes, mean acuity: 1.25, SE ± 0.047; 20/16) (Table 1). ARM in 16 of the 19 eyes of patients were classified as “dry” and three as “wet”. Six of 19 eyes in the ARM group had received intra-ocular lens implants during cataract surgery, which was also the case for 14 out of the 18 eyes of the age-matched control group. Since in most cases the implant causes mild myopia (−0.5 dpt), the patients-though presbyopic-could observe the fixation mark comfortably without near correction. Thus, optical artifacts from eyeglasses were prevented, such as aberrations or interference from the glasses’ rims, which would otherwise occur at higher eccentricities.
Table 1Age and visual acuity of the subject groupsAgeVisual acuity NMeanSEMinimumMaximumMeanSEARM group1973.792.360900.29 (20/63)0.048Age-matched controls1871.51.560780.65 (20/32)0.057Young controls827.11.721341.25 (20/16)0.047Visual acuity is described in decimal notation and Snellen fractions
Visual acuity (VA) was measured by a Landolt ring test (Binoptometer, Oculus, Germany) at 90 cm viewing distance (VA = 1/ω′, where ω′ is the minimum angle of resolution in minutes). Based on these values, all subjects were capable of directing their gaze at the fixation mark without glasses. All subjects with ARM and the age-matched control group provided an up-to-date medical statement from an ophthalmologist, which appraised the condition of the retina and excluded other eye diseases (e.g. glaucoma, secondary cataract). All subjects were examined by their ophthalmologist with respect to clarity of the optical media, and none of them showed any evidence of changes in the visual pathways.
The complete examination (including the initial interview) took approximately 3 hours.
The ages of the subjects with ARM and the age-matched controls were sufficiently close; a t-test with a preceding test for normal distribution showed that the two groups did not differ significantly with respect to their age (p = 0.36). The study design had been approved by the ethics committee of the University of Munich and testing procedures were in accordance with the tenets of the Declaration of Helsinki. All subjects gave their informed consent for participation.
Results
Motion contrast threshold
Figure 4 shows the contrast thresholds for motion perception as a function of eccentricity in the visual field. Compared with the age-matched controls, the ARM group shows a distinct increase of contrast thresholds up to 20° in the nasal and up to 40° in the temporal field. Mann-Whitney U-tests of the contrast thresholds at 10°, 20°, 30°, and eccentricity on the horizontal meridian each showed significant differences between the two elderly groups (Table 2).
Fig. 4Mean contrast thresholds for motion perception of the subjects with ARM, age-matched controls, and young controls on the horizontal meridian. The broken lines show the increased contrast thresholds when stimuli were presented near the blind spot. The continuous lines show the regression (specified in the formula); omitted for the age-matched control group on the temporal side for clarityTable 2Results of Mann-Whitney U-tests of contrast thresholds (ARM group vs. age-matched control group); U is the statistical value of nonparametric Mann-Whitney test for comparison of independent samples based on ordinal ranks; z  is the estimated statistical value of normalized distribution of U values; p  is the significance levelEccentricityNasal fieldTemporal fieldUzpUzp10°16.00−4.71<0.00153.00−3.59<0.00120°69.50−3.090.00272.50−2.830.00530°148.50−0.680.4996.00−2.280.02340°145.50−0.250.80102.00−2.100.03660°−−−134.00−1.120.261
Double-pulse resolution
With respect to double-pulse resolution, the subjects with ARM showed strongly increased thresholds compared with the age-matched controls within the 20° visual field covered by the test area (Fig. 5). For better comparison with the motion sensitivity data, Fig. 6 also shows the double-pulse results on the horizontal meridian. Both the comparison on the two horizontal meridians and on the entire circles at the investigated eccentricities (5°, 10°, and 20°) show significant group differences. We performed a Mann-Whitney U-test at every position on the horizontal visual field meridian (Table 3).
Fig. 5Mean double-pulse resolution (DPR) of the ARM group, age-matched controls and young controls at 5°, 10°, and 20° eccentricity (stimuli positioned on concentric rings, Fig. 3). The 0°-value represents the foveal DPR in the 5°-ring measurement. Note that the rather small SEM error bars indicate a limited range of variation of valuesFig. 6Double-pulse resolution (DPR) of the ARM group, age-matched controls, and young controls along the horizontal meridianTable 3Results of Mann-Whitney U-tests at each eccentricity for the ARM group and the age-matched control group; U is the statistical value of non-parametric Mann-Whitney test for comparison of independent samples based on ordinal ranks; z is the estimated statistical value of normalized distribution of U values; p is the significance levelEccentricityNasal fieldTemporal fieldUzpUzp5°46.50−2.550.01146.50−2.560.01110°45.00−3.83< 0.00179.50−2.780.00520°76.00−2.890.00361.00−3.340.001
Critical flicker fusion frequency (CFF)
Figure 7 shows critical flicker fusion frequency (CFF) as a function of age for the three subject groups. There is a slight but significant (p < 0.05) loss of CFF with age. It amounts to 0.059 Hz per year of age (i.e. around 5 Hz for the entire life span), which accounts for 30% of the inter-individual variance in the healthy subjects. Age-independent interindividual variance (70%) thus far exceeds the age-related loss. More importantly here, however, the graph also shows the marked loss of performance in the subjects with ARM. On average, the CFF in the ARM group is lower by 5.47 Hz, or approximately 14%, in comparison to the age-matched controls. Note that one 90-year-old subject reached the level of young normal subjects, while all other subjects with ARM showed lower performance than all young controls.
Fig. 7Critical flicker fusion frequency as a function of age for the three groups. The regression line refers to the healthy controls
The analysis of variance (one-way ANOVA with group as factor and post-hoc test, Tamhane procedure) indicates a highly significant difference between the three groups (F = 25.16; df = 2; p < 0.001) which was caused predominantly by the highly significant difference between the age-matched control group and the ARM group (p<0.001).
Color perception
Performance in color perception is a sensitive indicator of differential impairment of the parvo- vs. the magnocellular system [31]. The result from the Lanthony D-15 color vision test (unsaturated) allows the calculation of the color confusion score (CCS), which was used for the statistical analysis of the results. A one-way ANOVA with subject groups as factor and subsequent post-hoc test (Tamhane procedure) showed highly significant differences (F = 25.285; df = 2; p < 0.001) between the three test groups. The difference between the age-matched control group and the ARM group was also highly significant (p < 0.001). Thus, the subjects with ARM clearly showed impaired color discrimination. The comparison of crossings over the color space also shows significant differences between the subject groups (F = 20.394; df = 2; p < 0.001; mean crossings: ARM = 5.81, age-matched controls = 1.78, and young controls = 0).
Discussion
Motion perception
Our findings document deficits of dynamic visual field properties in ARM in a retinal region that extends far beyond the macula. In a sense, the well documented loss of visual function (e.g. foveal acuity) in the macula of subjects with ARM seems just like the proverbial tip of an iceberg. To our knowledge, this is the first study of dynamic characteristics of the peripheral visual field up to 60° eccentricity in subjects with ARM. Functional studies on these patients in the past have either concentrated on foveal function only, or have used a large central stimulus that did not discriminate between foveal and nonfoveal function [5, 40–42, 57]. Only the study by Brown and Lovie-Kitchen has shown deficits in the temporal visual field up to 20° eccentricity [13].
There are only a few studies on dynamic processing characteristics in ARM. Mayer et al. measured flicker sensitivity only foveally and suggest that it can be a predictor of exudative ARM [40, 41]. Normal aging of visual function – from which ARM losses need to be distinguished – has received only limited attention [19, 45, 46, 61].
In the present study, we determined the contrast thresholds for motion perception far into the periphery of the visual field on the horizontal meridian, up to 40° on the nasal, and 60° on the temporal side. We found pronounced impairment in the ARM group up to 20° nasally and 30° temporally, and smaller impairments still further out on the temporal side. The macula is commonly described as having a radius of 10° [2, 22]. These elevated contrast thresholds for motion perception extend far beyond this eccentricity, in conflict with a widespread assumption that the effects of manifest ARM are restricted to the macula. The latter seems to be based on two facts. First, customary descriptions and classifications of the disease use morphological features that are visible by ophthalmoscopy or on fundus photographs [6–8]; and second, many experimental studies have tested vision in subjects with ARM only in central locations in the visual field as noted above, with a few notable exceptions [10, 11, 55].
In clinical practice, the assessment of the visual periphery is believed to play a minor role in ARM, for a lack of consequences in ophthalmic treatment and also because of the dramatic vision loss from damage to the central retina, immediately noticed by patients who report the loss of the ability to recognize fine spatial or contrast detail (e.g. small print). A loss of peripheral motion sensitivity, in contrast, is not easily recognized by the patient, possibly because the conscious (sustained) component of attention is normally directed towards central vision, whereas the transient (reflex-like) component of attention is not accessible by consciousness [44]. This phenomenon plays a well-known role in glaucoma which often goes unnoticed by the patient. Thus, the fact that patients do not complain about deficits in peripheral motion perception might lead the practitioner not to examine this retinal region in detail. Yet, practical consequences have to be considered. If diminished sensitivity to motion raises the possibility that a patient’s transient attention might not be attracted to a moving stimulus, a behaviorally valuable eye movement may not be performed and a potentially hazardous situation may not be recognized.
Additionally, the tests available in routine clinical practice do not provide the appropriate stimuli to detect such defects. For instance, Holopigian et al. examined peripheral vision in patients with early ARM using standard electrophysiological and psychophysical tests (dark adaptation curves, electro-oculograms and electro-retinograms [23]. They found that only few subjects with early ARM developed disease-related effects in this region when examined with standard clinical tests.
Double-pulse resolution (DPR) and critical flicker fusion (CFF)
The results of DPR revealed distinct and statistically significant differences between the ARM group and age-matched controls up to 20° eccentricity. Furthermore, the increased foveal thresholds of double-pulse resolution in subjects with ARM parallel the elevated foveal CFF thresholds, which also show a dependency on age and a significant increase in subjects with ARM.
Comparison of the age-matched with the young control group shows an age dependency which confirms earlier results [18, 46, 48, 61]. However, our results cannot be directly compared with the findings of Falsini et al. [13] who used a large homogeneous test field (18° diameter) that covered visual field regions with pronounced differences in functional characteristics.
The fact that reduced dynamic performance in our patients was found for two quite different methods of measurement emphasizes the presence of impaired temporal vision performance far beyond the macula and suggests a common underlying pathogenesis.
Color vision
The objective of the color vision test was to examine the relative involvement of the two neuronal subsystems of the primary visual pathway – magnocellular (M) and parvocellular (P) – in the previously obtained results. Both motion perception and temporal resolution are assumed to be predominantly conveyed by the M system, whereas color vision is predominantly mediated by the P system [31]. To simply find out whether color perception is intact, it is legitimate to use the color vision test foveally, or to allow the subjects to use the retinal area that serves them best. A (seemingly self-evident) conclusion that the deficits reported here may be caused by a specific damage to the M system can thus be rejected. Impaired color vision, as found here, shows that the P system is also affected.
The ARM group in our study showed a significantly lower foveal color vision performance than the age-matched control group. This would seem in conflict with the results of Frennesson et al. and Medina et al. who found no color vision deficits in subjects with ARM at an early stage [14, 42]. However, those studies used less sensitive tests with saturated colors, such as the Farnsworth D-15 test or the Farnsworth-Munsell 100 Hue test, whereas our findings are based on the more sensitive Lanthony D-15 test which uses unsaturated colors [14, 42]. Since (static) contrast sensitivity was not measured here, we cannot estimate how strongly the color vision findings were influenced by the expectedly reduced contrast sensitivity of these patients.
Consequences
Even though the perceptual limitations measured in this study are not immediately evident to subjects with ARM, their relevance for daily life should not be underestimated. Their importance is due especially to the fact that segments of the near periphery assist safe mobility in general and driving a car in particular [21, 39]. Visual perception of moving stimuli has two functions of great importance in daily life. First, we perceive a stable, stationary world in which we are moving, despite the fact that nearly all image components on the retina are moving [43]. Being able to distinguish between the effects of self-motion and those of moving objects requires the detection and analysis of relative motion [47]. Second, our attention is constantly redirected to interesting and potentially dangerous objects, which typically cause a reorientation of gaze. These functions were examined previously in connection with the distinction between sustained and transient attention [3, 38, 44].
Because of the impairment of the central visual field in ARM, motion perception with peripheral vision is of even greater importance for these patients. Measurements with dynamic test stimuli are especially important for the assessment of driving ability of visually impaired persons [24]. Marron and Bailey examined contrast sensitivity and visual acuity with regard to their importance for orientation and mobility [39]. Their results provide evidence that contrast sensitivity and the integrity of the visual field are of considerably greater importance for safe mobility than visual acuity. Studies from our own group show that dynamic peripheral sensitivity in a divided attention condition was the strongest predictor for safe driving in a group of healthy elderly drivers [53]. Recent studies have confirmed the importance of peripheral vision for driving by finding that contrast sensitivity and visual field limitations are the strongest predictors for mobility of visually impaired persons [10, 21, 35]. These studies demonstrate the importance of the assessment of visually guided performance regarding moving objects and contrast vision. In the tests at hand, these two tasks are linked. The growing possibilities for mobility of the elder generation with age-related visual impairment require examinations that take into account seniors’ new lifestyles and can help ensure safe navigation.
Another relevant connection can be made with communication and the ability to read speech from the lips of a conversation partner, which is a difficulty faced by many elderly with additional hearing impairment. Although a general notion of the role of vision in speechreading has been established [56], the importance of motion sensitivity for speechreading has not been sufficiently investigated to date.

visual field
arm
temporal resolution
motion perception
contrast sensitivity