SUCCINIC ACID AND ITS DERIVATIVES: FERMENTATIVE PRODUCTION USING SUSTAINABLE INDUSTRIAL AGRO-FOOD BY-PRODUCTS AND ITS APPLICATIONS IN THE FOOD INDUSTRY

Headnote

ABSTRACT

The fermentative production of succinic acid (SA) is becoming an attractive alternative to traditional chemical synthesis. SA can be used directly or transformed into some derived chemicals, with applications in the food industry. For example, SA and sodium succinate are flavour enhancers; adipic acid and its salts can be used as acidity regulators, acidifiers, flavour enhancers, gelling aids and slow raising agents. Moreover, SA-derived biodegradable plastics, such as aliphatic polyesters and aliphatic-aromatic co-polyesters have a wide range of applications in food packaging. The replacement of a chemical process with a biological one offers many environmental advantages. To make it cost-competitive, however, process optimization is required and the substrate cost has to be reduced. To reach this goal, renewable low-cost resources, such as agro-food byproducts (molasses, cheese whey, wood hydrolysates, wheat flour) can be used in place of pure, expensive ones. In the present paper, the use of agro-food industry by-products as low cost carbon sources in the fermentative production of SA are reviewed. The use of SA and its derivatives in the food industry are also highlighted. Attention is focused on the innovative "cyclic" aspect of SA fermentative production and its applications from agro-food by-products to food products.

- Key words: agro-food by-products, biodegradable polymers, fermentation, food additives, succinic acid -

INTRODUCTION

The depletion of fossil fuel sources, increasing greenhouse gas emissions, climate changes and interest in using locally available, renewable raw materials have spurred the development of appealing bio-based processes to produce chemicals and fuels as an alternative to conventional petrochemicals. In many cases, biological processes are still more expensive than petrochemical ones (McKINLAY et al, 2007). Consequently, the key to a successful production of bio-based compounds is the development of an efficient, economically feasible manufacturing process.

Succinic acid (SA) is one of the most interesting and promising chemicals that can be successfully produced from renewable resources through a fermentation process (DU et al, 2007). At present, SA is produced through the hydrogenation of maleic anhydride, that is, using a petroleum source as the starting material (BECHTHOLD et al, 2008). Considering its wide range of potential chemical derivatives, its commercial value (LANDUCCI et al, 1994) and the potential market, the United States Department of Energy (DOE) has listed SA among the top twelve chemicals of greatest interest (WERPY and PETERSON, 2004). SA can be used directly or processed into many other value-added chemicals and commodities having several applications in various industrial fields (ZEIKUS et al., 1999; POTERA, 2005; SAUER et al, 2008). Therefore, SA is a so-called "building block chemical" or "platform chemical" . Focusing attention on the food industry, SA and its derivatives are used as food additives and for manufacturing biodegradable polymers.

In 2007, the total SA market and its derivatives amounted to 30 kton, of which 10% was used directly in the agricultural and food fields, 7% in SA-polymer production, 31% for coatings, pigment, dye and ink production and the remaining 52% was used in other fields of application (RUPP DAHLEM, 2008). The total market value for all of these SA applications is more than 400,000,000 US$/year (ZEIKUS et al, 1999). This value could potentially increase (US$ 1.3 billion /year) (http://www.wisbiorefine.org/ prod/sacid.pdf) thanks to the production of SA through microbial fermentation.

In order to develop a bio-based industrial SA production, that would be cost-competitive with traditional chemical processes, three main issues must be addressed: 1) the need to select low-cost raw materials; 2) the need to select high-yield producing microorganisms, that can metabolize a wide range of sugar feedstocks (WERPY and PETERSON, 2004); and 3) the need to reduce recovery and purification costs.

The price of raw materials greatly influences the overall process cost. The possibility of using agro-food industry by-products as low-cost carbon sources (C-sources) in the fermentative production of SA is described in this review and the promising future of SA building blocks is discussed.

Production of succinic

acid from microbial fermentation

SA is an intermediate in the TCA cycle (tricarboxylic acid cycle) in plants, animals and in many microorganisms. It is also one of the fermentation end-products of anaerobic microbial metabolism. Some microorganisms can also produce SA in aerobic fermentation through the glyoxylate pathway shunt (SANFACOR et al, 1976; McKINLAY et al, 2007).

Recent studies (LEE et al., 2002; LIU et al, 2008; MEYNIAL-SALLES et al, 2008) have focused attention on anaerobic rumen bacteria (e.g. Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens) because they are naturally able to produce and accumulate large amounts of SA (SONG and LEE, 2006). Furthermore, in the last few years, the use of recombinant strains has also been expanding (VEMURI et al, 2002a; LEE et al., 2006; KERN et al., 2007).

Currently, the fermentative production of succinic acid is generally carried out using defined carbon sources (glucose, fructose, xylose, saccharose, lactose, maltose) (VEMURI et al, 2002b; LEE et al., 2002; ISAR et al., 2006; LIU et al, 2008). However, some studies have demonstrated that agro-food by-products can be used as cheaper substrates, which would be important in a future perspective of an industrial scale production of SA.

Food by-products as low-cost substrates

Italian food industries produce an annual capital turnover of 1 13,000 billion euro; the main contributors are dairy products (14.2 billion euro), wine and liquor (10.7 billion euro), confectionary (10.1 billion euro), and fresh and processed meat (7.4 billion euro) (http://www.federalimentare.it/ Documenti/Cibus2008/Sottoprodotti.pdf). In addition to these main products, food plants and industries produce huge quantities of byproducts. According to Italian legislation (D. lgs. 152/2006), by-products are different from waste, in that they can be used to obtain value-added products. Their valorisation is a basic goal in a sustainable development strategy. Furthermore, exploitation of food by-products is an environmentally friendly approach which reduces their disposal as waste and could be an additional source of profit for industries themselves.

While SA-producing bacteria can use some by-products directly as a substrate (cheese whey and molasses), others (lignocellulosic materials and wheat flour) cannot be used directly because of their complex composition and structure. In the latter case, a two-step fermentative process is needed. In the first step, an inexpensive carbon source is converted into a stream of nutrients that are readily accessible to a variety of microorganisms; in the second step, the nutrient stream is converted into the desired product. The first step is usually a fungal bioprocess and the second is a bacterial one (DU et al., 2007).

To date, the fermentative production of SA has been reported for the following cost-competitive renewable feedstocks: cane molasses (0.073 US$/kg) (AGARWAL et al., 2006; NGUYEN and GHEEWALA, 2008), cheese whey (0.580.62 US$/kg) (WAN et al., 2008; http://usda. mannlib.cornell.edu/usda/current/DairProdPr/DairProdPr-07-10-2009.pdf), wood hydrolysates (0.025 US$/kg) (KAYLEN et al, 2000; KIM et al., 2004b; LEE et al., 2003a) and wheat flour (0.09-0. 134 US$/kg) (AKERBERG and ZACCHI, 2000; DU et al., 2007). The cost of a pure substrate (e.g. glucose, fructose, lactose or sucrose) is much higher: 40- 120 US$/kg (http://www. sigmaaldrich.com/sigma-aldrich/home.html).

It is crucial to take into consideration that the cost of each by-product can fluctuate appreciably, depending on: 1) the location of the agrofood plants and, consequently, on the availability of the by-product in a specific geographical area and 2) the annual production of the mainproduct. For example, the current price of cane molasses is 0, 196 US$/kg (http://www.agerborsamerci.it/listino/listino.html). All the abovementioned feedstocks will now be described in detail.

Cane molasses is a by-product from the sugar industry, with the following composition: total sugars such as fructose, glucose and sucrose (about 50%), suspended colloids, heavy metals, vitamins and nitrogenous compounds (ROUKAS, 1998). This by-product has been employed in "green" SA fermentation with A. succinogenes (LIU et al., 2008), Escherichia coli (AGARWAL et al., 2006) and Enterococcus flavescens (AGARWAL et al., 2007). A. succinogenes has proved to be the most efficient, giving the highest SA concentration (55.2 g/L) and productivity (1.15 g/L.h) in a fed-batch process (LIU et al., 2008).

Cheese whey is a major by-product in the manufacturing of cheese or casein from milk, and makes up about 80% of the volume of processed milk; it contains lactose (4.5-5% w/v), soluble proteins (0.6-0.8% w/v), lipids (0.4-0.5% w/v) and mineral salts (8-10% of dried extract). Cheese whey also contains appreciable quantities of other components, such as lactic and citric acids, non-protein nitrogen compounds (urea and uric acid) and group B vitamins (GONZALES SISO, 1996). In the past, whey was considered to be a pollutant because of its high Biological Oxygen Demand (BOD) (approximatively 45,000 mg/L). Hence, the disposal of huge quantities of this "waste" was an environmental and economic problem for dairy industries. At present, whey is recycled into useful products such as food ingrethents and animal feed or used for the recovery of value-added substances (such as lactose and proteins). The possibility of using cheese whey as a low-cost substrate is becoming a very appealing alternative.

Whey has been successfully fermented by E. flavescens (AGARWAL et al., 2007), A. succinogenes (WAN et al., 2008), A. succiniciproducens (SAMUELOV et al., 1999) and M. succiniciproducens (LEE et al., 2003b) to produce SA. A high concentration (34.7 g/L) and yield (90.6%) of SA were obtained using fed-batch fermentation by A. succiniciproducens; this strategy offers the advantages of both batch and continuous processes (SAMUELOV et al., 1999), M. succiniciproducens had the highest SA productivity (3.90 g/L.h) in a continuous mode (LEE et al., 2003b); A. succinogenes 130Z performed well in batch fermentation, producing 27.9 g/L of SA (WAN et al., 2008).

Lignocellulosic biomass can be obtained as a by-product from the agro-food industry. This substrate is not easily fermentated by bacteria but, considering the huge amount available, it is one of the most preferable inexpensive substrates. A two-step fermentative process is applied to a lignocellulosic biomass: in the first step, the lignocelluloses are hydrolyzed using a physical-chemical process (thermal, mechanical or acidic hydrolysis) or a biological one (treatment with ligninolytic fungal enzymes). In the second step, the cellulosic and hemicellulosic fractions are depolymerised to fermentable sugars (glucose and xylose) which can be used as substrate in microbial SA-fermentation (LEE, 1997). Unfortunately, some toxic or inhibitory compounds derived from the hydrolysis process are also present: this causes a decrease in the fermentative process yield and productivity (KIM et al., 2004b). These molecules (furfural, hydroxymethylfuran, p-hydroxybenzoic aldehyde and vaniline) (CONVERTI et al., 1998) can be removed but with a consequent increase in operational costs. Alternatively, an alkaline hydrolysate treatment is an effective and rather inexpensive method that reduces the formation of inhibitory compounds (KIM et al., 2004b).

Some researchers have reported fermentation by M. succiniciproducens and A. succiniciproducens in a wood-hydrolysed-based medium for SA production (LEE et al., 2003a; KIM et al., 2004b). A. succiniciproducens (LEE et al., 2003a) and M. succiniciproducens (KIM et al., 2004b) can metabolise wood hydrolysate for a "green" SA production with high SA yields (55-88% g SA/g substrate). The highest productivity (3.19 g/L.h) was obtained with continuous fermentation on a wood hydrolysate by M. succiniciproducens (KIM et al., 2004b).

As in the case of wood hydrolysates, SA can be produced in a two-stage bioprocess using wheat flour as starting material. In the first step, wheat flour is used as a substrate in fungal fermentation and converted into microbial feedstock. In the second step, the microbial feedstock (flour hydrolysate) is used in bacterial SA production (DU et al., 2007).

A. succinogenes is able to use flour hydrolysate (DU et al., 2007; 2008; LIN et al., 2008); the highest SA concentration obtained was 64.2 g/L and the yield was 0.81 g/g substrate, in a batch fermentation (DU et al., 2008).

The same microorganism can be efficiently used for SA fermentation in a wheat hydrolysate medium. A high SA concentration was produced (62,1 g/L) when a hydrolysate of a by-product of wheat flour milling was employed (DORADO et al., 2009).

The production of SA using food by-products and different bacterial strains in batch or fedbatch lab-scale fermentation are summarized in Table 1 where the working volume and the succinic acid concentration are reported.

Apart from substituting an expensive, pure C-source with a cheaper one, other expensive constituents of the medium can also be replaced with more economical ones. Some studies have reported the use of corn steep liquor (CSL), a byproduct of the corn wet milling industry (LEE et al., 2000; 2003b), or rice bran, a by-product of the rice-milling process (MOON et al., 2004), as low-cost nitrogen sources (N-sources) instead of complex, more expensive ones (peptone, yeast extract or trypton). These substrates also supply the medium with vitamins and minerals, in addition to proteins and amino acids. Quite comparable SA yields have been obtained in a fermentative process using both pure and cheaper N-sources (LEE et al., 2003a, b). If wood hydrolysate is used as the C-source, the SA yield is 88-89% (g/g) in the presence of polypeptone plus yeast extract and 85-88% (g/g) using CSL (LEE et al., 2003a). If whey is used as the C-source, the SA yield is 67-69% (g/g) in the presence of yeast extract and 65-66% in the presence of CSL (LEE et al, 2003b).

Potential applications of succinic acid

and its derivatives in the agro-food industry

Since SA is a linear saturated dicarboxylic acid, it can be used as an intermediate chemical. It can be converted into 1,4-butanediol (BDO), tetrahydrofuran (THF), γ-butyrolactone (GBL), and other four-carbon chemicals that have a world-wide market. For example, SA can be readily hydrogenated to 1 ,4-butanediol, which can then be further carbonylated to adipic acid (ZEIKUS et al., 1999) that is used as a food additive (see below).

Food additives

In Directive 95/2/EC (http://eceuropa.eu/ food/fs/sfp/addit_flavor/flavl l_en.pdf) on food additives which include colours and sweeteners, it is also possible to find SA, adipic acid and their salts. In the European Community, food additives are identified with a code composed of the letter E followed by a number. SA (E363) is a flavour enhancer or a buffer and neutralizing agent for food and beverages. Its applications and maximum level of use are reported in Table 2.

Sodium succinate can replace monosodium glutamate and dilysine succinate as a salty flavour enhancer for low-sodium food (ZEIKUS et al, 1999), Other potential applications of SA include: 1) the succinylation of lysine residues to improve the physical and functional attributes of soy proteins in food and 2) the use of succinylated starch as a thickening agent.

Adipic acid (E355) and its salts (sodium adipate E356 and potassium adipate E357) have different applications, which are reported in Table 2. Adipic acid is an acidity regulator, acidifier, flavour enhancer, gelling aid and a slow raising agent.

Starch sodium octenyl succinate (E 1450) and acetylated distarch adipate (E 1422) are used in weaning food or in infant formulas and followon formulas as emulsiflers, stabilizers, thickeners and bulking agents.

Succinic acid derived

biodegradable plastics

Of all the SA-derived chemicals, 1,4-butanediol and adipic acid are considered very important for the production of biodegradable polyesters and polyamides.

The Showa Denko company, produces 2 different series of aliphatic polyesters, under the trademark BIONOLLE. Bionolle1000 is a polybutylene succinate (PBS) constituted by 1,4-butanediol (BDO) and SA; Bionolle3000 is a polybutylene succinate adipate (PBSA), copolymer that is made by using 1 ,4-butanediol and a mixture of SA and adipic acid. Bionolle has some physical properties that are similar to traditional plastics (PE, PET) (MCCARTHY et al, 1999). Furthermore, thanks to its excellent processability, it can be processed into various moulded injected, extruded and blown products in conventional equipment commonly used for polyolefins (FUJUMAKI, 1998). The Bionolle series have a wide range of applications in the food sector: films (food packaging and wrapping films, bags, labels), lamination (paper lamination, bags, cups, containers) (KOBAYASHI et al, 2002), sheet extrusion (cards, can holders, food trays), blow moulding (drink bottles) and foam (cups and food trays). Bionolle can also be used coupled with starch to give blends that can be used in food packaging (GORMAL, 2002).

These SA polyesters are also manufactured under other trademarks: GS PIa (Mitsubishi Chemical), Skygreen (Sk chemicals), Lunare SE (Nippon Shokubai), EnPoI (IRe Chemicals Ltd). Different series, characterized by different physical properties, exist according to the required applications.

Polybutylensuccinate terephthalate (PBST) is produced by DuPont under the trademark Biomax. This material is usually tailored to mimic polyethylene or polypropylene. Biomax® resins are used for different applications, such as injection moulding for disposable cutlery, paper coating, thermoformable cups and trays, and certain films, such as lidding stock. This material possesses good barrier properties, which are among the best of all the biodegradable polymers. Biomax is mainly intended for final disposal through composting and in-soil degradation.

Polybutylenadipate terephthalate (PBAT) is an aliphatic-aromatic copolyester which is sold under the trade names Ecoflex (Basf) and EastarBio (Eastman). This copolyester combines the biodegradable properties of aliphatic polyester and the performance properties of aromatic polyesters. Two of the three monomers are constituted by 1,4-butandiol and adipic acid and can be obtained by using SA as the starting material. PBAT can be processed in different ways and then used for bags, cast films (agricultural mulching sheetings) and extrusion coating (paper coating, wrapping papers), fastfood packaging (drinking cups and hamburger boxes), food trays, disposable packaging for meat, fish, poultry, fruit and vegetables. It is biodegradable and compostable within about a maximum of 180 days. Consequently, these items can be eliminated organically, thus reducing their processing costs. PBAT looks and performs like low density polyethylene (LDPE). It is waterproof, sealable, printable and tearand temperature variation- resistant; it is also characterized by flexibility, anti-fogging performance and transparency.

The golden future

of the bio-based succinic acid platform

The fermentative production of SA is a very promising way for expanding the use of SA and its derivatives on the market. Taking into account the potential use of agro-food by-products as low-cost substrates, the fermentation approach becomes more interesting from an economic and environmental point of view.

The economic interest is associated with the transformation of the by-products used as raw materials into value-added products and the reduction in the costs of by-product disposal.

The environmental advantage is gained by replacement of petrochemical resources with renewable ones, such as feedstocks, the fixation of carbon dioxide in microbial fermentation (lower emissions of greenhouse gases) and replacing traditional plastics (non-degradable) with SA-derived biodegradable ones.

From an economic point of view and that of process bottlenecks, consideration must be given not only to the cost of raw materials, but also the cost of the downstream process. The recovery and purification steps are difficult and crucial. In fact, an industrial agro-food by-product has SA in the medium but also large amounts of additional undesirable substances. These substances must be removed, resulting in an increase in the overall process costs.

At present, three methods are used for the downstream process: electrodialysis (GLASSNER and DATTA, 1992), acidification (DATTA et al, 1992) and solvent extraction (KIM et al., 2004a). These methods however cannot be applied on an industrial level to recover and purify SA because they are too costly. Furthermore, they must be improved in order to obtain purer SA. A new method has recently been set-up that consists of reactive extraction, vacuum distillation and crystallization (HUH et al., 2006). It should noted that all of these methods were first developed in culture broth containing pure C-sources. A great deal of effort is necessary to set up and optimize the downstream process that uses a culture medium that contains complex C-sources, such as agro-food by-products.

The low cost of raw materials having a complex substrate is offset by the need to remove the toxic or inhibitory molecules (e.g. wood hydrolysate-derivatives) during the downstream steps (GLASSNER and DATTA, 1992; YEDUR et al, 2001; HUH et al., 2006). Moreover, all the fermentation products derived from bacterial metabolism, other than those from SA, must also be purified.

The main goals of the SA production process are: to optimize the culture conditions and fermentation parameters, to use engineered microorganisms, to increase SA yield and productivity, to reduce by-products formation and to set up a better performing SA recovery method.

"Green" SA and its derivatives make up a more "natural" food-additive category, that can be used instead of traditional chemical ones. Biodegradable plastics produced from "green" SA are also an attractive alternative in the food packaging sector. Both these aspects could have a successful impact on public opinion.

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Revised paper received July 30, 2009 Accepted October 30, 2009