Title

Process for the Production of Succinic Acid

Field of the Invention The invention relates generally to succinic acid production. More specifically, the invention relates to producing succinic acid using fermentative microbes.

Background of the Invention

Sustainable production of fuels and chemicals is driven by the need to control atmospheric concentrations of greenhouse gases such as carbon dioxide, depletion of existing oil reserves and increasing oil prices. Production of bio-based environmentally benign fuels and chemicals has been hindered by the increased cost versus fossil fuel based alternatives. Compared to oil refineries, biotechnical processes must be integrated to utilize raw materials efficiently. For example, in the manufacture of bioethanol, a significant percentage of the carbon in the biomass is discharged as carbon dioxide.

Although this carbon dioxide is from biological sources and thus not environmentally harmful, it still compromises the process economics.

Some attempts have been made to solve this problem by integrating ethanol plants with other biotechnical processes in order to better utilize raw materials. The structure of such a plant is similar to an oil refinery and is termed a biorefmery [1, 2]. One drawback with biorefmeries is that they efficient users of carbon. For example, in the production of ethanol for fuel from biomass, one half of the mass of incoming carbon is diverted to carbon dioxide, resulting in a process with only a 50% yield of ethanol based on carbon source. It would improve process economics if the ethanol yield could be increased and/or if the diverted carbon dioxide could be used.

Succinic acid is considered to be one of the top twelve chemical building blocks that potentially can be manufactured from biomass [3]. Succinic acid can be used to derive a wide range of commodity and specialty chemicals. Examples include diesel fuel additives, deicers, biodegradable polymers and solvents, and detergent builders. Carbon

dioxide is used in the production of succinic acid giving the process a negative net release of the greenhouse gas. Since the process utilizes carbon dioxide it can be appropriate to connect it to an ethanol plant in order to recover the carbon that would otherwise be lost through the release of carbon dioxide.

Succinic acid can be produced by a number of organisms including Bacteroides ruminicola and Bacteroides amylophilus, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes and Escherichia coli. Bacteroides ruminicola and Bacteroides amylophilus can produce succinic acid in high yields, but the organisms have a tendency to lyse after rather short fermentation times [4]. A. succiniciproducens presents problems in handling since it is an obligate anaerobe. In addition, its medium needs to be supplemented with tryptophan and the final titer is only about 35-40 g/L [5, 6]. A. succiniciproducens produces acetic acid in a ratio of 2: 1 succinic acid to acetic acid. Actinobacillus succinogenes strain 130Z has been shown to produce succinic acid in concentrations of 80-110 g/L [7]. Although very high, these concentrations are obtained using a medium supplemented with biotin and yeast extract. Such nutrients are expensive and therefore not suitable when applying the technology to large scale production. E. coli is well known, easy to handle and can produce the desired acid in high yield when sugars are fed slowly and when there is a large amount of water supplied throughout the process [5]. By keeping sugar availability very low, metabolic overflow and the production of excess acetic acid is avoided. Unfortunately, the use of low sugar concentrations to avoid metabolic overflow results in a high dilution of the fermentation broth which increases the downstream costs of product recovery. Furthermore, the low sugar availability results in slow volumetric productivity that in turn causes increased capital costs for fermentation vessels.

The choice of microorganism is related to three main considerations when developing a bio-based industrial production process for succinic acid. First, a low cost medium must be used. Second, the producing organism must be able to utilize a wide range of sugar feedstocks in order to make use of the cheapest available raw material. The third and most important factor for producing succinic acid economically is volumetric

productivity [3]. Previous work has reported 1 g/L/h [5, 8]. To make succinic acid production feasible, productivity in the range of 2-2.5 g/L/h will be necessary [3].

In 1949 J. L. Stokes demonstrated that E. coli under anaerobic conditions produces a mixture of organic acids [9]. Typical yields from such a fermentation is 1.2 moles formic acid, 0.1-0.2 moles lactic acid, and 0.3-0.4 moles succinic acid per mole glucose consumed. To use E. coli in a process for producing succinic acid, the yield of succinic acid relative the other acids had to be increased. In that regard, different E. coli mutants have been developed by the U.S. Department of Energy (USDOE). Two such mutants are AFPl 11 and AFP 184. AFPl 11 is a spontaneous mutant with mutations in the glucose phosphotransferase system, the pyruvate formate lyase system and in the fermentative lactate dehydrogenase system [6]. The mutations result in increased succinic acid yields and were deliberately inserted in a near wild type strain, the C600 (ATCC Accession Number 23724), which can ferment both five- and six-carbon sugars and which has strong growth characteristics [5].

E. coli mutants have been developed which possess the ability to produce succinate under aerobic conditions [10, 11]. Obtained yields are at the maximum for aerobic fermentation (1 mole succinate per mole glucose), but productivity is low, only about 1 g/L/h [10, 11, 12]. Recombinant AFPl 11 and strains with similar mutations have been used to overexpress pyruvate carboxylase; yields obtained were in the range of 1.4 - 1.5 moles succinic acid per mole glucose [13, 14]. Another strain with deletions of ldhA, adhE, ack- pta, iclR, and overexpressing pyruvate carboxylase was able to produce succinic acid with yields in excess of 1.7 moles succinate per mole glucose [15]. One thing all of these studies have in common is the use of rich media, moderate sugar concentrations, supplementation with antibiotics, and a maximum productivity of approximately 1 g/L/h. As mentioned above, for commercial application of microbial succinic acid production from biomass it is essential to have high productivity in order to make it competitive with petroleum based alternatives. The high productivity should ideally result from a process that can be based on multiple types of minimal media and which makes conservative use of, or avoids completely, additional resources such as water, antibiotics, and nutrient

supplements. There remains an unmet need in the art to provide such commercially- viable processes for the microbial production of succinic acid.

Summary of the Invention It is therefore an object of the present invention to provide a process for succinic acid production which can utilize known organisms and minimal media but which produces high yields of succinic acid. It has surprisingly been found that AFP 184 can be grown in a minimal media to produce succinic acid from glucose, fructose, xylose, and mixtures of glucose and fructose and glucose and xylose with productivities in the range of 1.5 - 2.9 g/L/h.

According to a first embodiment of the invention, a process for producing succinic acid is provided which comprises supplying a media with E. coli AFP 184 and at least 50 g/L sugar under aerobic conditions, subsequently converting the media to anaerobic conditions, and harvesting succinic acid from the media. The sugar can be a five carbon sugar or a six carbon sugar, and it can be selected from the group consisting of: glucose, fructose, xylose, a mixture of glucose and fructose, and a mixture of glucose and xylose. For example glucose which could be supplied at a concentration of at least 100g/L to the process for producing succinic acid. An antifoam agent can also be supplied during the aerobic conditions step. The process can be conducted within the range of 35-40 ⁰ C and/or within the range of pH 6-8. For example the temperature could be approximately 37 ⁰ C and the pH could be approximately 6,8. Aerobic conditions can persist for at least 24 and up to 48 hours, for example, from 6-8 hours. The aerobic conditions can persist until optical density of a sample of media reaches 550 nm. Anaerobic conditions could persist for 24-48 hours, for example, 16 hours.

According to a further embodiment of the invention, a process for production of succinic acid is provided which comprises providing a sterile media, adding >50g/L sterile sugar solution and E. coli AFP 184 to the media, fermenting the media under aerobic conditions for at least 4 hours, converting the media to anaerobic conditions and fermenting the media under anaerobic conditions for at least 8 hours, and harvesting the succinic acid.

According to a further embodiment of the invention, a device for producing succinic acid is provided which comprises means for containing a fermentative microorganism in a media, means for supplying sugar to the media at a concentration of at least 50 g/L, means for oxygenating the media to create aerobic conditions, means for flushing the media with carbon dioxide to create anaerobic conditions, and means for withdrawing succinic acid from the media.

According to a further embodiment of the invention, a biorefmery is provided which comprises means for producing ethanol, and means for producing succinnic acid according to the process described above.

Brief Description of the Drawing Figures

Figure 1 shows sugar concentrations in g/L as a function of time for Glucose (circles),

Fructose (diamonds), Xylose (triangles), where the vertical lines symbolize the transition to the anaerobic phase;

Figure 2 shows sugar concentrations in g/L as a function of time for Glucose (Glucose :Fructose fermentation, circles), Fructose (diamonds), Glucose (Glucose :Xy lose fermentation, x's), and Xylose (triangles), where the vertical lines symbolize the transition to the anaerobic phase; and

Figure 3 shows a mixed acid fermentation pathway for AFP 184, the effects of the mutations in the pfl system and the ldh system are represented by broken lines.

Detailed Description

It has surprisingly been found that E. coli strain AFP 184 can be utilized in dual-phase fermentation using high initial sugar concentrations. The first fermentation stage is an aerobic growth stage and the second stage is an anaerobic production phase. The dual- phase fermentation process can be run in batch mode and/or continuous mode.

For example, initial sugar concentrations of 50-100 g/L, where the sugar is glucose, fructose, xylose, mixtures of glucose and fructose, or mixtures of glucose and xylose can be used. Alternatively, the initial sugar concentration could be 50-200 g/L. The process can be carried out within the temperature range of approximately 30-40 ⁰ C and with pH values of approximately 6-8.

The aerobic growth phase can last approximately 2 to 8 hours, or until optical density reaches about 30-40. The anaerobic production phase can last approximately 24-48 hours, or until the sugar is depleted.

The process is optimized when measurements of relevant parameters such as oxygen concentration and viable cell count are taken as necessary and any useful adjustments are made throughout the process. The resulting productivity is surprisingly high, at least 1.5 g/L/h. Ranges using approaches described herein are expected to be approximately 1.5 - 3 g/L/h. Further developments in the field may allow for even higher yields when using the inventive method.

When converting the media to anaerobic conditions the flow of air is stopped and the flow of carbon dioxide starts. Harvesting is initiated after the fermentation process is completed for removal of the product i.e. the succinic acid from the media solution. The succinic acid produced can be removed from the mixture after removal of cells according to known techniques such as electrodialysis, crystallization, ion exchange, or esterification followed by distillation.

While the invention is applicable to independent production of succinic acid, it is particularly well-suited to be performed in conjunction with biotechnical processes such as the manufacture of bioethanol. Because biotechnical processes can result in a net excess of carbon dioxide, it is preferred to have a complementary process which utilizes that excess carbon instead of releasing it into the environment. When used in connection with glucose, the process optimizes the conversion of carbon dioxide into succinic acid because the starting materials provide a 6 + 2 carbon source and the product, succinic

acid, is a 4 carbon molecule. This optimizes the process dynamics by providing a carbon supply that is evenly divisible by the number of carbons in the molecule produced, a preferred stoichiometry.

The present invention will now be described with specificity in accordance with certain of its preferred embodiments; however, the following examples serve only to illustrate the invention and are not intended to limit the same. If sources are not specifically described materials and equipment are known and commercially available.

Preparation of E. coli AFP184 Inoculum

E. coli strain AFP 184 lacks functional genes coding for pyruvate formate lyase, fermentative lactate dehydrogenase, and the glucose phosphotransferase system [5]. Cultures were obtained from the USDOE (also available from American Type Culture Collection, Manassas, Virginia, USA, ATCC Accession Number 202021) and stored at -80 ⁰ C as 30% glycerol stocks. The inoculum was prepared by inoculating four 500 mL shake-flasks containing 125 mL sterile Tryptone Soy Broth (TSB) medium with 200 μL of the glycerol stock culture. The inoculated flasks were incubated for 16 h at 37°C (200 rpm).

Fermentation

Fermentations were conducted in a 12 L fermenter (BRl 2, Belach Bioteknik AB, Sweden) with a total starting volume of 8 L (including 0.5 L inoculum). The medium used (developed at Oak Ridge National Laboratories, Oak Ridge, TN, USA) contained the following components per 8 L: 11.2 g of K2HPO4; 4.8 g of KH2 PO4; 26.7 g of (NH4)2SO4; 1.6 g of MgSO4 and 266g of Corn Steep Liquor (50% solids).

The medium was sterilized in the fermenter at 121 ⁰ C for 20 min; thereafter 2 L sterile sugar solution (400 g/L), 3 mL antifoam agent (Antifoarn 204, SigmaAldrich) and 500 mL inoculum were aseptically added. During the whole fermentation the temperature was controlled at 37°C and the pH was maintained at 6.7 by automatic addition of a 15% NH3

solution. The dissolved oxygen concentration (%DO), measured by a pO2 electrode, was kept above 30% by varying the agitation speed.

Total fermentation time was 24 hours and consisted of an aerobic growth phase and an anaerobic production phase. During the aerobic growth phase (6-8 h) the culture medium was aerated with an air flow of 10 L/min. After 8 hours, or when the optical density at 550 nm had reached a value of 35, the anaerobic production phase was initiated by withdrawing the air supply and flushing the culture medium with CO2 at a flow rate of 3 L/min. Succinic acid was produced during this anaerobic production phase which continued for the remaining 16 hours of the fermentation. During fermentation, samples were withdrawn aseptically for analysis of optical density, viable cells, sugars and organic acids concentrations.

Cell Growth Cell growth was monitored by measuring the optical density at 550 nm (OD550). Since optical density measures not only living cells but also other solids in the media (including dead cells), it was only used as a reference for when the anaerobic production phase should be initiated (results not shown). To establish the number of viable cells, ten-fold serial dilutions of the fermentation samples were plated on tryptone soy agar plates and grown overnight at 37°C. The number of colonies was then calculated and the number of viable cells was expressed as colony forming units (CFU) per milliliter fermentation broth.

Organic Acid and Sugar Analysis The concentrations of organic acids were measured by HPLC (Series 200 Quaternary LC pump and UV-VIS detector, PerkinElmer) equipped with a Cl 8 column (Spherisorb, 5μm, 4.6mm x 150mm, Waters) using a 50 mM KH2PO4 buffer with 2% acetonitrile at a flow rate of 0.35 mL/min as the mobile phase. Samples for acid analysis were centrifuged at 10,000 rpm for 10 minutes at 4 ⁰ C. The supernatant was diluted with the mobile phase and filtered through a 0.22 μm syringe filter.

Sugar concentrations were determined using the same HPLC system as above, but equipped with a Series 200 Refractive index (RI) detector (PerkinElmer), guard column and an ion exchange column (Aminex HPX87-P, BioRad). The column was kept at 85°C in a column oven for optimal performance. Prior to analysis, the samples were centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was diluted with water and filtered through a 0.45 μm syringe filter. Water at a flow rate of 0.6 mL/min was used as the mobile phase. All data was collected and processed using PerkinElmer' s TotalChrom analytical software. Peak areas from the chromatograms were evaluated through comparison to standard curves prepared from solutions with known concentrations of succinic acid, acetic acid (4, 2, and 1 g/L), fructose, xylose and glucose (10, 5, 2.5 and 1 g/L). In this way, fermentations with glucose, fructose, xylose, equal mixtures of glucose and fructose, and equal mixtures of glucose and xylose were performed and analyzed. The succinic acid produced can be removed from the mixture according to known techniques. Comparisons of the different sugar feedstocks on succinic acid production follow.

Single Sugar Fermentations

Glucose- and fructose-based fermentations gave approximately the same growth rate during the first six hours, but fermentations using glucose as the sole carbon source resulted in a higher concentration of viable cells at the switch to anaerobic conditions. As shown in Table 1 , the glucose fermentation resulted in a higher final concentration of succinic acid than the fructose or xylose fermentations.

Apart from succinic acid, small quantities of acetic acid were also produced. The ratio of acetic acid to succinic acid produced was highest in the fructose fermentation. The best succinic acid yield was obtained in the glucose fermentation and was 0.92 gram succinic acid per gram glucose consumed in the anaerobic phase. The yield from the fructose fermentation corresponded to 50% of the yield from the glucose fermentation and the yield from the xylose fermentation was 0.69 gram succinic acid per gram xylose consumed in the anaerobic phase.

By running the fermentations with starting sugar concentrations of 100 g/L, the system had a high concentration of sugar available when the switch to the anaerobic phase was made (Fig. 1) and therefore the productivity was not limited by the carbon source. The highest productivity for single sugar fermentations, 2.8 g/L/h, was obtained from the glucose fermentation. The fructose fermentation resulted in the lowest productivity, 1.5 g/L/h. The productivity of the xylose fermentation was somewhat higher than the productivity of the fructose fermentation, reaching 1.8 g/L/h. The sugar consumption rates in the glucose, fructose and xylose fermentations were almost equal (Fig. 1).

Mixed Sugar Fermentations

Fermentations were conducted with an equal mixture of glucose and fructose and glucose and xylose, respectively. The glucose: fructose fermentation resulted in the highest concentration of viable cells, even higher than in the fermentation using glucose as the sole carbon source. As with the glucose fermentation, this mixed fermentation also produced a succinic acid concentration above 45 g/L (Table 1). The productivity in the glucose:fractose fermentation was very high, 2.9 g/L/h. The acetate concentration reached 8 g/L and the ratio of succinic acid to acetic acid was thus lower than for the glucose fermentation, but higher than for the fructose fermentation. The yield of the mixed glucose and fructose fermentation is just between the yields obtained using either sugar as sole carbon source (Table 1). During the mixed glucose: fructose fermentation all sugars were consumed (Fig. 2). As mentioned above, this fermentation also resulted in the highest succinic and acetic acid concentrations, but not the highest yields. Table 1 discloses, succinic and acetic acid concentrations, mass yields in grams succinic acid per gram sugar and molar yields in moles succinic acid per mole sugar at the end of the fermentation after 24 hours and productivity during the anaerobic phase. Yields are based on the amount of sugar consumed in the anaerobic phase. Table 1

Parameter Glucose Fructose Xylose G lucose : Eructose Glucose:Xylose

Succinic add (g/L) 45.4 27.7 29.2 52.0 31.2

Acetic acid (g/L) 5.6 6.3 5.1 8.0 3.4

Yield (g/g) 0.92 0.46 0.69 0.73 0.57

Yield (mole/mole) 1.41 0.69 0.88 1.12 0.78

Productivity (g/L/3i) 2.84 1.54 1.79 2.89 1.73

The glucose :xylose fermentation had a viable cell density and final succinic acid concentration in the same range as the fermentations using only fructose or xylose. It also resulted in a similar productivity. However, the final acetic acid concentration was low, only 3.4 g/L. As a result, the glucose:xylose fermentation had the highest ratio of succinic acid to acetic acid. Interestingly, the yield was actually higher in the case of a pure xylose fermentation than with a mixture of glucose and xylose. In the mixed glucose :xy lose fermentation, the xylose is utilized faster than the glucose (Fig. 2).

As noted by way of introduction, there is a need in the art to provide organisms and processes which product succinic acid in an industrially-applicable way. To this end, the present invention provides a fermentation process which relies on both aerobic and anaerobic phases and for which multiple sugars can be used. The process of the present invention produces succinic acid in commercially- viable yields while at the same time remaining sufficiently flexible to accommodate a variety of starting materials. This makes efficient use of raw materials and reduces production costs because the process can tolerate variations in the composition of the feed and still obtain a good conversion of the raw materials into the desired product.

Previous studies had only reached yields of 1 g/L/h [6.5 6] whereas the present invention leads to production of approximately 3 g/L/h, a three-fold increase. High productivity was in part due to high starting sugar concentration. This inventive finding has demonstrated a novel solution to previously-reported drawbacks when using large amounts of sugar, i.e., metabolic overflow resulting in undesired acetic acid production.

In addition to high starting sugar concentration, fermentations were run in fed-batch mode, adding a sugar solution at low flow rate in order to keep the system limited with

respect to sugar. Thus, it has now been surprisingly found that it is possible to subject AFP 184 to high sugar concentrations under anaerobic conditions without any additional acetate production compared to fed-batch fermentations. Since the process has sugar available at the onset of the anaerobic phase, it was possible to increase the productivity and reduce the production times to about one third as compared to similar fermentations performed in fed-batch mode and still obtain the same final succinic acid concentration.

Once the new process for increasing productivity without negative effects was discovered, the utilization of different carbon sources could be investigated as described in detail, above. This provides further guidance to skilled workers who choose to rely on the novel process for succinic acid production and who want to optimize the same. From the time needed to reach the desired optical density, growth on fructose was more rapid than growth on glucose. This result may be due to the mutation in the phosphotransferase system (PTS) of AFPl 84.

When glucose is mixed with other sugars it is usually consumed first and the other sugars later, a phenomenon called catabolite repression [18]. From the sugar consumption rates (see Figs. 1 and 2) it was observed that the glucose and fructose were consumed at approximately the same rate. This is an expected effect of the AFP 184 mutation in the glucose PT system. Without the mutation the PTS would phosphorylate the glucose first. The highest yield was obtained when glucose was used as the sole carbon source. The yield of succinic acid observed in the 100% fructose fermentation was half of the yield observed in the 100% glucose fermentation. Again, without being bound by theory, the way the sugar is phosphorylated by the cells may be the cause.

In AFP 184, glucose is phosphorylated from ATP by the enzyme glucokinase, while fructose is phosphorylated from phosphoenolpyruvate (PEP) by the PT system [22, 23]. Phosphoenolpyruvate is also a node in the mixed acid fermentation pathway. A schematic picture of the mixed acid fermentation pathways of E. coli, including the effects of the mutations in AFPl 84, is shown in Figure 3. From PEP, oxaloacetate is formed by carboxylation. The reactions then proceed through malate, fumarate and finally succinate.

When fructose is used as the carbon source, one molecule of PEP is used for fructose phosphorylation yielding pyruvate from which only acetate can be formed. As an result, only half the amount of succinic acid can be produced from the same amount of sugar. Increasing the percentage of glucose in the mixture increases the yield linearly towards the yield of 100% glucose fermentation. From a redox balance the maximum yield of succinate per mole of glucose is 1.714 or 1.12 g succinate/g glucose [13]. The yield obtained from a pure glucose fermentation was 1.4 moles succinate/mole glucose, which is 82% of the theoretical max. In E. coli glucose can also enter the cell via mannose- specific permeases of the PTS. The mannose- specific permeases also utilize PEP to phosphorylate the sugar [19, 16, 18, 24]. Since the permeases are not affected by the mutation in the ptsG gene it is also possible for AFP 184 to phosphorylate glucose with these permeases. Hence some of the PEP will be lost as pyruvate, which to some extent can explain the yield deviating from the theoretical.

In fermentations based solely on glucose or xylose the sugars are consumed at the same rate (Fig. 1), but in the fermentation with mixtures of glucose and xylose, xylose is consumed faster (Fig. 2). The yield is lower for xylose-containing fermentations. Considering stoichiometry, the yield from xylose must be lower than the yield from glucose. Interestingly, the succinic acid yield in the mixed glucose :xy lose fermentation was lower than the yield from the fermentation based only on xylose. This could be due to xylose being used faster and to a larger extent than glucose. Acetic acid production was lower in the xylose fermentations than in the glucose or fructose fermentations.

The viable cell density was determined by cultivation on agar plates. For the pure fructose fermentation the number of viable cells was almost constant between 8 and 24 hours. The same observation was made for the mixed glucose :xylose fermentation. The pure xylose fermentation demonstrates an increase in the viable cell concentration during the anaerobic phase. A common factor in all three of these fermentations was that the succinic acid concentration was around 30 g/L. In the glucose and mixed glucose: fructose fermentations a large decrease in the number of viable cells was observed. The final succinic acid concentration in these fermentations was in the range of 45-50 g/L.

Fermentations were run for longer than 24 hours with more sugar added after 24 hours, but the viable cell density continued to decrease and no more succinic acid was produced (results not shown). In a lean media AFP 184 cannot tolerate succinic acid concentrations of more than 45-50 g/L. The effects observed can be due to toxicity or a product inhibition affecting, for example, the activity of some essential metabolic enzyme.

Thus, the present invention presents a new process for using an E. coli strain in a dual- phase fermentation with five- and/or six-carbon sugars to produce succinic acid with anaerobic productivities in the range of 1.5-3 g/L/h and yields of as high as 1.4 moles of succinic acid per mole of sugar consumed anaerobically. The invention provides improved opportunities for utilizing bio-based succinic acid production in a commercially- viable way.

The foregoing disclosure has been set forth merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.

Numerals in parentheticals throughout the application text generally refer to specific references, the details of which are reproduced below. All of the following are incorporated by reference in their entirety.

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