Effective production of succinic acid from coconut water (Cocos nucifera) by metabolically engineered Escherichia coli with overexpression of Bacillus subtilis pyruvate carboxylase.

Succinic acid is an important acid which is used in medicine and pharmaceutical companies. Metabolically engineered Escherichia coli strain was used for the effective production of succinic acid using Cocos nucifera water, which contained 5.00 ± 0.02 g/L glucose, 6.10 ± 0.01 g /L fructose and 6.70 ± 0.02 g /L sucrose. Fermentation of C. nucifera water with E. coli M6PM produced a final concentration of 11.78 ± 0.02 g/L succinic acid and yield of 1.23 ± 0.01 mol/mol, 0.66 ± 0.01 g/g total sugars after 72 h dual-phase fermentation in M9 medium while modeled sugar was 0.38 ± 0.02 mol/mol total sugars. It resulted in 72% of the maximum theoretical yield of succinic acid. Here we show that novel substrate of C. nucifera water resulted in effective production of succinic acid. These investigations unveil the importance of C. nucifera water as a substrate for the production of biochemicals.

Introduction

Succinic acid is a platform chemical that has been identified as one of the top building block chemicals [1,2]. Succinic acid and its derivatives have many applications in medicine, chemical, pharmaceutical, agriculture, polymers and plastics industries, with a potential market of $15 billion [3]. The future economic and environmental profits of a bio-based succinate manufacturing have driven investigation and improvement of succinate-producing organisms. [3]. There are numerous biotechnological companies utilizing metabolic engineered microorganisms to manufacture succinic acid, for example Reverdia using Saccharomyces cerevisiae [4], Myriant using Escherichia coli [5], Bioamber using Pichia kudriavzevii [6] and Succinity using Basfia succiniciproducens [7]. Presently, succinic acid production by hydrogenation of the petroleum-derived maleic anhydride is expensive for utilization. Inexpensive microbial production of succinic acid from sugars has emerged over the past two decades.

E. coli is a facultative anaerobic gram-negative microorganism and effective in the utilization of carbon sources for heterotrophic growth [8]. It is acknowledged as an excellent microorganism due to its rapid growth, simple genetic manipulation and recognized metabolism [[9], [10], [11]]. E. coli can produce succinic acid during aerobic and anaerobic processes [12,13]. During the anaerobic processes, it utilizes the reductive branch of the tricarboxylic acid (TCA) pathway function in E. coli as the key pathway for the production of succinic acid. However, during the aerobic processes succinic acid is produced utilizing the whole oxidative TCA cycle. The anaerobic process for the production of succinic acid is more profitable [14], since it can guarantee a high yield of the main chemical because of the CO2 fixation at the stage of oxaloacetate (OAA) formation, the main precursor metabolite of the reductive branch of the TCA pathway [15]. Other microorganisms which can produce succinic acid includes Anaerobiospirillum succiniciproducens [16], Mannheimia succiniciproducens [12], Corynebacterium glutamicum [17], Actinobacillus succinogen [18], B. succiniproducens [19], S. cerevisiae [20] and Yarrowia lipolytica [21].

Cocos nucifera (coconut tree) belong to the palm tree family (Arecaceae) [22,23]. Coconut water (coconut liquid endosperm), is very useful and multipurpose natural product which makes it to be abundant [24]. Due to its abundance in the region where it is cultivated and grown the liquid may be wasted. It is a revitalizing beverage that is naturally disbursed worldwide because it is beneficial and valuable for health. Coconut water is conventionally utilized for plant tissue culture or micropropagation [25]. The comprehensive uses of coconut water can be acceptable by its distinctive chemical composition of sugars, vitamins, minerals, amino acids and phytohormones [26]. Hence we utilized C. nucifera water for the production of succinic acid due to its importance, abundance and may be wasted. Cocos nucifera water is a wastewater originating from the southeastern part of Asia. About 200,000 tons of C. nucifera water was produced in Thailand in 2001 [27]. It can be collected free of charge when there is available transportation for the waste. It can be used for the production of sports drinks and food gels. The major sugars that can be derived from it include glucose, fructose, and sucrose [28]. Purified C. nucifera water from Brazil showed elevated reductase activity at ambient temperature in aliphatic, aromatic aldehydes and ketones, which indicates that C. nucifera water investigation for the synthesis of organic compounds is still underexplored [29]. Investigation of C. nucifera water is deprived, mainly focusing on specific uses, biochemical compositions, and preservation techniques. Therefore, in this investigation genetically engineered strain was constructed and established for succinic acid production from C. nucifera water. Three major components of C. nucifera water (glucose, fructose, and sucrose) were investigated for the production of succinic acid in the dual-phase fermentation.

Materials and methods

Strains and plasmids

Strains, plasmids and primers used in this study are presented in Supplementary Table 1. Wild-type E. coli K-12 strain MG1655 and all the genetic modifications were performed on the strain or derivatives. The pyruvate carboxylase (pyc) gene was amplified from Bacillus subtilis and plasmid-pTrcHisA pyc was developed using restriction enzymes Kpn I and Hind III. Here, we use catAsacB selection marker for the gene deletion, which is better than λ-red recombinase and group II introns retrotransposition which leave scars in the genome and limits allelic exchange application. Forward and reverse primers contained 50 bp homologous sequences upstream or downstream of the target gene followed by 25 bp sequences corresponding to the chloramphenicol acetyltransferase and levansucrase cassette (catAsacB). The DNA fragment was amplified from peasycatAsacB and subjected to electroporation of E. coli MG1655 or derivatives carrying pKD46 for homologous recombination. The resistant strain catAsacB and target gene deleted was screened and verified by colony PCR and sequencing of the DNA. The catAsacB was then removed by culturing in 10 ml LB medium in a 50 ml beaker overnight (around 12 h). The next day the 1 ml of the cultures were inoculated into 10 ml LB medium with 10% sucrose, 0.5% yeast and 1% tryptone. After grown to the late stationary phase, the cultures were streaked on an LB agar. The grown colonies were then transferred with and without chloramphenicol simultaneously. Finally, colonies, which lost the chloramphenicol resistance, were screened for the catAsacB removal [30].

Media

All modified strains were grown in LB medium (per liter): 10 g tryptone, 5 g yeast and 10 g NaCl. Other supplements include 100 mg/mL ampicillin, 50 mg/L kanamycin and 34 mg/mL chloramphenicol. M9 minimal medium contained 800 mL H2O, with 64 g Na2HPO4-7H2O, 15 g KH2PO4, 2.5 g NaCl, and 5.0 g NH4Cl. The solution was stirred until all salt dissolved and adjusted to 1000 mL with distilled H2O and sterilized in an autoclave. 700 mL distilled H2O (sterilized), 200 ml of M9 salts, 2 mL of 1 M MgSO4 (sterilized), 20 ml of 20% glucose (or other source of carbon) and 100 μL of 1 M CaCl2 (sterilized) was added and the solution adjusted to 1000 mL with distilled H2O.

Cloning and overexpression of the gene pyruvate carboxylase (pyc)

The plasmid pTrchisA was used as the template for cloning the pyc gene with the trc and its promoter. The pyc gene was amplified with primers pyc-F and pyc-R (Table 1). Both primers 4 μM, synthesized at Sangon Company Beijing China were mixed with 10 ng template, 4 μL dNTPs and 0.5 μl Taq DNA polymerase and sterile water to form a 50 μL PCR mixture all from Beijing Trans Gen Biotech Company China. The PCR conditions were 94 °C for 4 min., 94 °C for 20 s, 60 °C for 20 s, 72 °C for 1 min. with a final step of 72 °C for 8 min. for 35 cycles. The PCR was confirmed using the agarose gel electrophoresis and PCR products were extracted using the Axygen (Axygen Scientific Inc). The pTrchisA was extracted and digested using Kpn I and Hind III overnight and constructed using gibson assembly mix. The plasmid was then transformed into E. coli to generate the strains shown in Table 1. The strains were grown in 100 ml LB medium at 37 °C by adding 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

Table 1

Dual-phase fermentation of the different engineered strains after 48 h in M9 medium using 20 g/L glucose.

Strains	DCW a (g/L)	Glucose consumed a (g/L)	Fermentation products a (g/L)				Yield a (mol/mol)
			Succinate	Pyruvate	Acetate	Formate
MG 1655	0.58 ± 0.01	18.00 ± 0.03	1.08 ± 0.07	0.13 ± 0.01	5.00 ± 0.06	0.32 ± 0.01	0.08 ± 0.01
MGG	0.47 ± 0.01	17.00 ± 0.02	0.44 ± 0.05	0.25 ± 0.03	4.00 ± 0.09	N.D	0.04 ± 0.02
M6P	0.62 ± 0.06	17.00 ± 0.02	2.65 ± 0.01	0.58 ± 0.02	N.D	N.D	0.23 ± 0.01
M6PM	0.54 ± 0.05	19.00 ± 0.01	1.82 ± 0.05	0.68 ± 0.01	N.D	N.D	0.15 ± 0.01

a Each values is the mean of three parallel replicates ± standard deviation.

b ND not detected.

Yield was calculated as mol succinate produced per mol glucose metabolized.

Dual-phase fermentation on pure glucose in the shaking flasks

M9 medium was used for aerobic and anaerobic fermentation. The medium was supplemented with glucose, 100 mg/L ampicillin, and 50 mg/L kanamycin. 1 mL of seed inoculum from an overnight 10 mL culture was added into 250 mL flask containing 50 mL M9 medium for aerobic growth at 37 °C and 200 rpm agitation speed. Preliminary optimization of transitional time anaerobic fermentation was carried out after 6 h of aerobic incubation (OD600 = 2.0). 10 g/L glucose, 1 g MgCO3, and 0.2 mM IPTG were placed in an anaerobic incubator (CIMO Medical Instrument Manufacturing Ltd., Shanghai, China) without shaking at 37 °C for 48 h. Samples at 0 h and 48 h in anaerobic phase were collected and stored for analysis of biomass and glucose.

Dual-phase fermentation of a modeled sugar mixture and C. nucifera water in 5 L- bioreactor by engineered E. coli M6PM

Dual-phase fermentation of modeled sugar mixture of C. nucifera water was carried out in 5-L bioreactor (New Brunswick Scientific Co., Inc. USA) containing 2 L M9 medium. In aerobic phase, 5% seed cultures were inoculated. The flow rate of sterile air was controlled at 5 L/min with 350 rpm of agitation speed with the temperature kept at 37 °C. The anaerobic phase was started when OD600 reached 2.0, CO2 gas was sparged with a flow rate of 2 L/min and agitation speed decreased to 150 rpm. Simultaneously, the concentrated mixture was gradually fed into the medium. The pH was kept at about 7.0 with 10 M NaOH and 10% H2SO4. The dual-phase fermentation of C. nucifera water was performed using the same method as described above. Sugars from C. nucifera water were supplemented at the transition time from aerobic phase to anaerobic phase. All bioreactor fermentation experiments were performed in triplicate.

Analytical methods

The OD600 was determined to monitor cell growth and correlated to the dry cell weight (DCW): DCW (g/L) = 0.36 × OD600. Glucose, fructose, sucrose and fermentation products (succinate, acetate, formate, and pyruvate) were quantified by high-performance liquid chromatography (HPLC) equipped with UV absorbance detector and refractive index detector (Agilent Technologies, USA). The Aminex HPX-87H ion-exchange column was used for analysis (Bio-Rad, USA) and operated at 50 °C with a mobile phase of 5 mM H2SO4 solution at a flow rate of 0.6 mL/min.

The yield of succinic acid was calculated with the following formula respectively:

Results and discussion

Dual-phase fermentation of the different engineered strains using glucose

During the dual-phase fermentation of the different engineered strain using glucose only about 5% of the maximum theoretical yield of succinic acid was produced in the wild-type E. coli while the metabolic flux was directed into the production of byproducts. It could be seen vividly that during the deletion of only the 6-phosphogluconate dehydrogenase (gnd) there was no significant difference from the wild-type (Table 1). This shows that the deletion of only the gnd is not effective for the production of succinic acid because there is still formation of byproducts which is not important for the production of succinic acid. The 6-phosphogluconate dehydrogenase (gnd) is involved in the production of ribulose 5-phosphate and functions in the pentose phosphate pathway as the main generator of cellular NADPH. It is located in the boundary of oxidative and non-oxidative pentose phosphate pathway. Our result for the MGG in which only gnd was deleted is in consonant with previous research with titer value of 0.35 g/L succinic acid of using glucose [31].

Hence the byproducts pathways were deleted by the inactivation of the ldhA, pta-ackA, poxB, pflB, pgi, and down-regulation of ppc with overexpression of pyc (Fig. 1). There was a significant increase in synthesis of succinic acid by 13.4% maximum theoretical yield compared to the wild-type (Table 1). This demonstrates that genetically engineering of the pathway may enhance the yield of succinic acid production.

Fig. 1

Production of succinic acid using modeled sugar in 5 L bioreactor.

Dual-phase fermentation of the different engineered strains using fructose

Fructose which is one of the major sugars present in the C. nucifera water was utilized as a substrate for the production of succinic acid. Fructose can be referred to as a C6 polyhydroxyketone. Table 2 shows that after 48 h dual-phase fermentation on 20 g/L fructose, the concentrations of succinic acid in the wild-type E coli MG 1655, MGG, M6P and M6PM were 1.08 ± 0.08 g/L, 1.57 ± 0.05 g/L, 1.75 ± 0.01 g/L, and 2.00 ± 0.05 g/L respectively. Utilization of carbon source for the production of succinic acid in the MGG alone is not sufficient to disallow the accumulations of undesired products. Hence mutation, down-regulation and overexpression were done to enhance the synthesis of succinic acid.

Table 2

Dual-phase fermentation of the different engineered strains after 48 h in M9 medium using 20 g/L fructose.

Strains	DCW a (g/L)	Fructose consumed a (g/L)	Fermentation products a (g/L)				Yield a (mol/mol)
			Succinate	Pyruvate	Acetate	Formate
MG 1655	0.58 ± 0.01	18.00 ± 0.03	1.08 ± 0.07	0.13 ± 0.01	5.00 ± 0.06	0.32 ± 0.01	0.08 ± 0.01
MGG	0.48 ± 0.01	19.00 ± 0.02	1.57 ± 0.05	0.29 ± 0.03	4.00 ± 0.09	N.D	0.13 ± 0.01
M6P	0.42 ± 0.02	10.00 ± 0.02	1.75 ± 0.01	0.60 ± 0.02	N.D	N.D	0.27 ± 0.01
M6PM	0.47 ± 0.03	12.20 ± 0.01	2.00 ± 0.05	0.78 ± 0.01	N.D	N.D	0.30 ± 0.01

a Each values is the mean of three parallel replicates ± standard deviation.

b ND not detected.

Yield was calculated as mol succinate produced per mol fructose metabolized.

Influence of sucrose substrate on dual-phase fermentation of the different engineered strains

Gene mutation, down-regulation of phosphoenolpyruvate carboxylase and overexpression of Bacillus subtilis pyruvate carboxylase in E. coli MGG, M6P and M6PM enhanced the production of succinic acid by 170%, 145% and 67%, respectively and thus shows the efficiency of the gene mutation and overexpression of the Bacillus subtilis pyruvate carboxylase gene utilizing surcrose (Fig. 3). Here, the percentage increase of succinic acid production during the deletion of 6-phosphogluconate dehydrogenase (gnd) during the utilization of sucrose may be due to the fact that the pentose phosphate pathway has been known to be major routes of sugar catabolism, generating reducing equivalent and production of cellular metabolites for cellular activities [32]. Little is known about the metabolism of sucrose in the central metabolic pathway if gnd is deleted in the pentose phosphate pathway. Hence, blockage of the PPP may redirect the metabolic flux and activate the alternative mechanism. 6-phosphogluconate dehydrogenase (gnd) is located between the oxidative and non-oxidative pentose phosphate pathway which may have induced activation of the pyruvate carboxylase (Fig. 3). Therefore, deletion of only gnd could lead to the accumulation of 6-phosphogluconate during dual-phase fermentation stemming from the inability of the oxidative pathway to function effectively, and may also reduce the functions of ED pathway (Table 3).

Fig. 3

Succinic acid production from recombinant E.coli using sucrose, fructose, and glucose.

NAD+, oxidized nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NADH, reduced nicotinamide adenine dinucleotide; NADP+ oxidized nicotinamide adenine dinucleotide phosphate. Red star for knock out arrow green for overexpression and yellow triangle for knock down. lactate dehydrogenase A (ldhA), phosphotranacetylase acetate kinase A (pta-ackA), pyruvate formate lyase B (pflB), pyruvate oxidase B (poxB), phosphoglucose isomerase (pgi), Glucose phosphotransferase system (ptsG), pyruvate carboxylase(pyc), phosphoenol pyruvate carboxykinase (pck), phosphoenol pyruvate carboxlyase (ppc), galactose permease (galP), phosphofructose kinase A (pfkA), fructose bisphosphate aldolase(fba), malate dehydrogenase (mdh), fumarase (fum), formate hydrogen lyase (fhl) glucose-6-phosphate dehydrogenase (zwf),6-phosphogluconate dehydrogenase (gnd), 6-phosphogluconolactonase (pgl).

Table 3

Dual-phase fermentation of the different engineered strains after 48 h in M9 medium using 20 g/L sucrose.

Strains	DCW a (g/L)	Sucrose consumeda (g/L)	Fermentation productsa (g/L)				Yielda (mol/mol)
			Succinate	Pyruvate	Acetate	Formate
MG 1655	0.58 ± 0.01	18.00 ± 0.03	1.08 ± 0.07	0.13 ± 0.01	5.00 ± 0.06	0.32 ± 0.01	0.08 ± 0.01
MGG	0.54 ± 0.01	17.00 ± 0.04	2.92 ± 0.05	0.28 ± 0.03	4.00 ± 0.09	N.D	0.27 ± 0.01
M6P	0.54 ± 0.02	10.00 ± 0.02	2.65 ± 0.01	0.52 ± 0.02	N.D	N.D	0.40 ± 0.01
M6PM	0.64 ± 0.01	18.20 ± 0.01	1.80 ± 0.05	0.77 ± 0.01	N.D	N.D	0.15 ± 0.01

a Each values is the mean of three parallel replicates ± standard deviation.

b ND not detected.

Yield was calculated as mol succinate produced per mol sucrose metabolized.

Dual-phase fermentations of the different engineered strains

C. nucifera water is composed mainly of fructose, glucose and sucrose, which makes it an important substrate for the production of succinic acid. Firstly, dual-phase fermentation of MP6M strain was investigated after 48 h in M9 medium with C. nucifera water in shaking flasks. The C. nucifera water and sugar mixture was added into the fermentation medium with about 17.8 g/L initial total sugars. It could be seen that MP6M could consume 5.20 ± 0.02 g/L glucose, 6.00 ± 0.02 g/L fructose and 6.00 ± 0.03 g/L sucrose in the modeled sugar which produced a final yield of 0.30 ± 0.01 mol/mol of succinic acid. E.coli M6PM consumed all the sugars in the C. nucifera water completely within 48 h of fermentation and produced a final yield of succinic acid of 0.43 ± 0.02 g/g (Table 4) which was slightly better than the modeled sugar. It was favored than that of M6P and MGG which were 0.37 ± 0.03 g/g (Table 5) and 0.28 ± 0.02 g/g (Table 6) respectively. Hence E. coli M6PM was selected for dual-phase fermentation in the 5 L bioreactor.

Table 4

Dual-phase fermentations of E. coli M6PM after 48 h in M9 medium with different carbon sources.

Medium	DCW a (g/L)	Glucose consumed a (g/L)	Fructose consumed a (g/L)	Sucrose consumed a (g/L)	Fermentation products a (g/L)					Yield a (g/g)
					Succinate	Pyruvate	Acetate		Formate
Medium A	0.60 ± 0.01	5.20 ± 0.02	6.00 ± 0.02	6.00 ± 0.03	5.08 ± 0.02	0.13 ± 0.01	4.00 ± 0.06	0.32 ± 0.01		0.30 ± 0.01
Medium B	1.69 ± 0.01	5.00 ± 0.01	6.10 ± 0.01	6.70 ± 0.04	7.64 ± 0.03	0.25 ± 0.03	4.00 ± 0.09	0.22 ± 0.01		0.43 ± 0.02

Yield was calculated as succinate produced (g/L)/ glucose consumed g/L + fructose consumed (g/L) + sucrose consumed (g/L).

Medium A represents the sugar mixture (Glucose, fructose and sucrose).

Medium B represents C. nucifera water.

Table 5

Dual-phase fermentations of E.coli M6P after 48 h in M9 medium with different carbon sources.

Medium	DCW a (g/L)	Glucose consumed a (g/L)	Fructose consumed a (g/L)	Sucrose consumed a (g/L)	Fermentation products a (g/L)					Yield a (g/g)
					Succinate	Pyruvate	Acetate		Formate
Medium A	0.68 ± 0.01	5.20 ± 0.02	6.00 ± 0.02	6.00 ± 0.03	5.00 ± 0.02	0.53 ± 0.01	4.00 ± 0.06	0.32 ± 0.01		0.29 ± 0.01
Medium B	1.67 ± 0.01	5.00 ± 0.01	6.10 ± 0.01	6.70 ± 0.04	6.54 ± 0.03	0.65 ± 0.03	4.00 ± 0.09	0.22 ± 0.01		0.37 ± 0.03

Yield was calculated as succinate produced (g/L)/ glucose consumed g/L + fructose consumed (g/L) + sucrose consumed (g/L).

Medium A represents the sugar mixture (Glucose, fructose and sucrose).

Medium B represents C. nucifera water.

Table 6

Dual-phase fermentations of E. coli MGG after 48 h in M9 medium with different carbon sources.

Medium	DCW a (g/L)	Glucose consumed a (g/L)	Fructose consumed a (g/L)	Sucrose consumed a (g/L)	Fermentation products a (g/L)					Yield a (g/g)
					Succinate	Pyruvate	Acetate		Formate
Medium A	0.60 ± 0.01	5.20 ± 0.02	6.00 ± 0.02	6.00 ± 0.03	4.08 ± 0.02	0.13 ± 0.02	5.00 ± 0.02	2.52 ± 0.01		0.24 ± 0.01
Medium B	1.60 ± 0.01	5.00 ± 0.01	6.10 ± 0.01	6.70 ± 0.04	5.04 ± 0.03	0.20 ± 0.01	6.00 ± 0.05	2.60 ± 0.01		0.28 ± 0.02

Yield was calculated as succinate produced (g/L)/ glucose consumed g/L + fructose consumed (g/L) + sucrose consumed (g/L).

Medium A represents the sugar mixture (Glucose, fructose and sucrose).

Medium B represents C. nucifera water.

Utilization of glucose, fructose, and sucrose for succinic acid production using E. coli M6PM during dual-phase fermentation

In Fig. 1, the modeled sugar was prepared with 5.20 ± 0.02 g /L glucose, 6.00 ± 0.02 g/L fructose, and 6.00 ± 0.03 g/L sucrose. The dry cell weight (DCW) was 1.15 ± 0.01 g/L, which yielded 3.76 ± 0.02 g/L succinic acid. The modeled sugar produced 0.38 ± 0.02 mol/mol total sugar after 72 h dual-phase fermentation. E.coli MP6M consumed 5.20 ± 0.02 g/L glucose, 6.00 ± 0.02 g/L fructose and 6.00 ± 0.03 g/L sucrose effectively within 36 h, 48 h and 72 h respectively. The average sugar consumption rates were 0.14 ± 0.01 g L−1  h-1, 0.13 ± 0.01 g L−1  h-1 and 0.08 ± 0.01 g L−1  h-1, respectively, which revealed that both glucose and fructose was consumed faster than sucrose. The catabolic repression of glucose on non-preferred carbon sources may be responsible for the initial consumption of glucose [[33], [34], [35]].

Effective production of succinic acid from C. nucifera using E. coli M6PM

In order to verify the fermentation attributes of C. nucifera water as the substrate for the production of succinic acid dual-phase fermentation was carried out in a 5 L bioreactor (Fig. 2). Hence, 17.8 g/L total sugars were used in the C. nucifera investigation. E. coli M6PM exhibited different consumption of sugars from the modeled sugar. The three sugars were consumed simultaneously during the fermentation. Glucose was consumed more rapidly than fructose and sucrose. Glucose was consumed finally after 36 h while sucrose and fructose were utilized finally after 48 h. This may be due to the fact that murein cluster C gene (mreC) which is responsible for the rod shape [36,37], has been deleted, which allow the accumulation of succinic acid. The increased cell growth (1.7 ± 0.02 g/L DCW) may be as a result of the presence of good nutrient in the C. nucifera water which may enhance the production of succinic acid [28].

Fig. 2

Production of succinic acid using Cocos nucifera water in 5 L bioreactor.

Fermentation of C. nucifera water with E. coli M6PM produced a final concentration of 11.78 ± 0.02 g/L succinic acid and yield of 1.23 ± 0.01 mol/mol, 0.66 ± 0.01 g/g total sugars after 72 h dual-phase fermentation in M9 medium. This makes up 72% of the maximum theoretical yield and is comparable to some results of succinic acid production in the literature [[38], [39], [40], [41], [42]]. Hence, C. nucifera water could be used for the production of succinic acid in large scale and other biochemicals. Therefore, metabolically engineering E. coli M6PM (MG1655ΔldhAΔptaackAΔpoxBΔpflBΔpgiΔmreCppc pyc), may result in a better molar yield of succinic acid using C. nucifera water by enhancing the strain design.

Conclusions

E. coli M6PM was developed for succinic acid production using C. nucifera water which utilized the substrate wastewater effectively for the production of succinic acid. It produced 11.78 ± 0.02 g/L succinic acid and a yield of 1.23 ± 0.01 mol/mol, 0.66 ± 0.01 g/g total sugars after 72 h dual-phase fermentation in M9 medium, which resulted in 72% of the maximum theoretical yield. Hence, this work put forward that C. nucifera water could be used for the production of succinic acid and other biochemicals in industrial scale.

Declaration of Competing Interest

The authors declare no conflict of interest