Bio-based Succinic Acid Production Research Articles

Sustainable succinic acid production from lignocellulosic hydrolysates by engineered strains of Yarrowia lipolytica at low pH

Succinic acid (SA) is a valuable C4 platform chemical with diverse applications. Lignocellulosic biomass represents an abundant and renewable carbon resource for microbial production of SA. However, the presence of toxic compounds in pretreated lignocellulosic hydrolysates poses challenges to cell metabolism, leading to inefficient SA production. Here, engineered Yarrowia lipolytica Hi-SA2 was shown to utilize glucose and xylose from corncob hydrolysate to produce 32.6 g/L SA in shaking flasks. The high concentration of undetoxified hydrolysates significantly inhibited yeast growth and SA biosynthesis, with furfural identified as the key inhibitor. Through overexpressing glutathione synthetase encoding gene YlGsh2, the tolerance of engineered strain to furfural and toxic hydrolysate was significantly improved. In a 5-L bioreactor, Hi-SA2-YlGsh2 strain produced 45.34 g/L SA within 32 h, with a final pH of 3.28. This study provides a sustainable process for bio-based SA production, highlighting the efficient SA synthesis from lignocellulosic biomass through low pH fermentation.

Reconfiguration of the reductive TCA cycle enables high-level succinic acid production by Yarrowia lipolytica

Succinic acid (SA) is an important C4-dicarboxylic acid. Microbial production of SA at low pH results in low purification costs and hence good overall process economics. However, redox imbalances limited SA biosynthesis from glucose via the reductive tricarboxylic acid (TCA) cycle in yeast. Here, we engineer the strictly aerobic yeast Yarrowia lipolytica for efficient SA production without pH control. Introduction of the reductive TCA cycle into the cytosol of a succinate dehydrogenase-disrupted yeast strain causes arrested cell growth. Although adaptive laboratory evolution restores cell growth, limited NADH supply restricts SA production. Reconfiguration of the reductive SA biosynthesis pathway in the mitochondria through coupling the oxidative and reductive TCA cycle for NADH regeneration results in improved SA production. In pilot-scale fermentation, the engineered strain produces 111.9 g/L SA with a yield of 0.79 g/g glucose within 62 h. This study paves the way for industrial production of biobased SA.

Recent Advancements and Strategies of Improving CO2 Utilization Efficiency in Bio-Succinic Acid Production

The production of bio-based succinic acid through microbial CO2 fixation and conversion has gained significant attention as a promising approach to mitigate greenhouse gas emissions. However, the low CO2 utilization efficiency limits the efficient biosynthesis of succinic acid. Therefore, it is crucial from environmental and economic perspectives to enhance the efficiency of CO2 utilization in bio-succinic acid production. This review comprehensively covers the introduction of biosynthetic pathways for microbial CO2 fixation and the conversion of CO2 to succinic acid, as well as the challenges associated with CO2 supply and utilization effectiveness. Moreover, strategies including genetic and metabolic engineering for CO2 fixation, extracellular supply methods of CO2 and some potential technical approaches for CO2 capture (such as micro-nano bubbles, CO2 adsorption material and biofilm) are summarized and presented.

Bio-based succinic acid production from durian husk: A rising Southeast Asia agricultural waste

The spiking availability of durian husk in Southeast Asia reveals a promising yet underutilized lignocellulosic biomass. Compositional characterization via National Renewable Energy Laboratories protocols (NREL) showed that durian husk contained 40.96% glucan, 9.51% xylan, and 26.48% of lignin. Enzymatic hydrolysis of durian husk was improved upon dilute sulfuric acid pretreatment. The optimal conditions for particle size and pretreatment duration were determined to be − 80/+ 100 mesh size durian husk exposing to an hour of acid pretreatment, resulting in a maximum reducing sugar recovery of 37.29 and 24.32 g/L for acid and enzyme hydrolysate, respectively. Fermentability tests using Actinobacillus succinogenes 130Z for both acid and enzyme hydrolysates demonstrated succinic acid yields of 49% and 63% (g/g), respectively. A stoichiometry equation describing the formation of substrate and all products was established based on the results obtained from batch fermentation: C6H12O6 + 0.377 C5H10O5 + 0.130NH3 + 0.238CO2 → 0.864 C4H6O4 + 0.224 C2H4O2 + 0.134CH2O2 + 0.292CH2.096O0.111N0.658 + 0.231 H2O. This equation serves as a useful reference for future investigations into succinic acid production reactions. The cost-effectiveness of succinic acid production and its environmental performance were evaluated to highlight the potential of durian husk as feedstock. This study presents the first report on the utilization of durian husk as an alternative and sustainable feedstock for the production of bio-based succinic acid.

Succinic acid: applications and microbial production using organic wastes as low cost substrates

Abstract Succinic acid is a valuable organic acid with a high commercial value that may be employed in a variety of sectors including food, cosmetics, and chemistry. Through bacterial fermentation, succinic acid can be easily produced. This paper includes a broad body of literature assessment spanning the previous two decades on the evaluation of succinic acid (SA) production procedures in to further drive research toward membrane-based sustainable and affordable production. The best natural method of SA producer is through Actinobacillus succinogenes. The process of microbial fermentation is used to produce bio-succinic acid utilizing agro-industrial waste. There are different methods under metabolic engineering which are being frequently used for bio-based succinic acid production using representative microorganisms, such as Mannheimia succiniciproducens, Pichia kudriavzevii, Saccharomyces cerevisiae, Actinobacillus succinogenes, Corynebacterium glutamicum, Basfia succiniciproducens, and Escherichia coli. This review summarizes the evolution of microbial production, fermentative methods, various organic substrates and the effects of efforts to recover and refine components for a wide range of applications in the perspective of biologically produced succinic acid for commercialization state.

Enhancing Succinic Acid Production by Sequential Adaptation of Selected Basfia succiniciproducens Strains to Arundo donax Hydrolysate

Promising green technologies that can overcome the challenges associated with the use of fossil fuels require microorganisms that can effectively ferment lignocellulosic hydrolysate for biochemical production with reduced sensitivity to toxic chemicals derived from the pretreatment process. In this study, a sequential adaptation approach was developed to obtain new bacterial lines from Basfia (B.) succiniciproducens strains, which are adapted to inhibitory compounds of the Arundo (A.) donax hydrolysate or those that accumulate during the fermentation process. The early adaptation stages resulted in newly adapted B. succiniciproducens bacterial lines that can tolerate fermentation end-products such as acetic, lactic, and succinic acids, as well as toxic compounds such as furfural and hydroxymethylfurfural. These adapted bacterial lines were further investigated to assess their ability to produce succinic acid in an MHM medium supplemented with a filtrate of A. donax hydrolysate. Batch growth tests on a small laboratory scale showed that bacterial lines 2E and 4D produced 5.80 ± 0.56 g L−1 and 5.81 ± 0.39 g L−1 of succinic acid, respectively, after 24 h of fermentation. Based also on its growth rate, the adapted bacterial line B. succiniciproducens 4D was selected for tests in a lab-scale fermenter, where it was able to synthesize up to 17.24 ± 0.39 g L−1 of succinate (corresponding to YSA/gluc 0.96 ± 0.02 g g−1 and to YSA/(G + X) 0.48 ± 0.01 g g−1) from MHM medium added with A. donax hydrolysate. Experiments showed an increase of ~17% compared to the control strain. The overall results demonstrate the potential of adapted bacterial lines for succinate production from A. donax hydrolysate and the development of improved technologies for bio-based succinic acid production.

Succinic Acid Production from Glycerol by Actinobacillus succinogenes: Techno-economic, environmental, and exergy analyses

A significant commercial chemical namely succinic acid can be produced using glycerol from the biodiesel industry. Previously, key attributes of the upstream and the downstream processing in the synthesis of bio-based succinic acid were identified, which substantially influence the technological competitiveness compared to petroleum-based production. Accordingly, this study aims to simulate succinic acid production from glycerol with the most promising technology. The techniques of reactive extraction and direct crystallization, as well as the use of dimethyl sulfoxide (DMSO) as an electron acceptor, are investigated. Selected scenarios are evaluated based on techno-economic, energy, exergy, and greenhouse gas (GHG) emissions performances to acquire a broader perspective on the sustainability of the technology. The obtained results indicate that the addition of DMSO in the fermentation is key for the bio-based succinic acid production – the best profit of 190 million USD of the net present value, 33.3% of the internal rate of return, and 4.48 years of the payout period are estimated. Additionally, bio-based succinic acid production could compete with the petrochemical route, particularly the reduction of GHG by 26%. Therefore, the production of succinic acid from glycerol is feasible given the positive net present value and lower GHG emissions compared to the petrochemical route.

Evaluation of a Prototype for Electrochemical pH-Shift Crystallization of Succinic Acid.

Downstream processing of biotechnologically produced carboxylic acids, such as succinic acid, poses environmental and economic challenges. Conventional downstream processes cause large amounts of waste salts, which have to be purified or disposed of. Therefore, lean and waste-free downstream processes are necessary for the biotechnological production of succinic acid. Electrochemical downstream processes gain especially significant attention due to low chemical consumption and waste reduction. This work presents the pH-dependent solid-liquid equilibrium of succinic acid, a prototype for electrochemical pH-shift crystallization processes, and its characterization. Based on the supersaturation, energy consumption, and electrochemical protonation efficiency the proposed electrochemical pH-shift crystallization is evaluated. This evaluation highlights the potential of the proposed electrochemical crystallization processes as waste-free and economically attractive processes for bio-based succinic acid production.

The design and techno economic analysis of a succinic acid production facility

Succinic acid is an industrially important commodity chemical that acts as a precursor to numerous other chemicals. This article explores an alternative biochemical route (as opposed to the traditional route from petroleum), by modeling a succinic acid production plant using Actinobacillus succinogenes, to convert the renewable sugars of corn stover feedstock. The model developed with SuperPro Designer v9.0 was used to evaluate the feasibility of this approach. This design is on par with the throughput of currentlarge scale manufacturers, by achieving an annual production rate of 11.5 US kilotons of succinic acid. The plant's economic standing is very attractive with a payback time of just under 5 years and return on investment of 20.4%. This article shows a facility design that maximizes profitability for a feasible and sustainable solution for bio-based succinic acid production.

Inhibition kinetics of bio-based succinic acid production by the yeast Yarrowia lipolytica

Kinetic models that describe the fermentative SA production by the yeast Yarrowialipolytica were developed for the first time. A modified Monod model considering both substrate and product inhibition based on shake flask experiments is proposed to describe the cell growth. Y. lipolytica tolerated up to 302.4 g/L glycerol, and significant decrease in SA yield and prolonged lag phase were observed with glycerol concentration above 212.0 g/L. Among the end-products investigated, acetic acid (AA) was found to have the most inhibitory effect on SA fermentation. The critical concentrations of SA and AA were 243.4 g/L and 57.9 g/L respectively. These demonstrated the greater potential of the yeast than bacterial SA producers. An unstructured model, based on a basic mass-balance of carbon, was used to describe substrate consumption, from which the cell maintenance coefficient ms was estimated to be 0.118 g/(g h). The Luedeking–Piret model was applied to describe the accumulation of organic acids, with SA and AA production following a mixed-growth-associated pattern. The values of the growth-associated term of both SA (0.386 g/g-DCW) and AA (0.208 g/g-DCW) were much higher than those of the non-growth-associated term of SA (0.013 g/(g-DCW h)) and AA (0.010 g/(g-DCW h)) respectively, indicating these products were mainly formed in tandem with yeast growth. After optimisation and validation, the model simulations match the experimental observations well, enabling elucidation of the fermentation characteristics for Y. lipolytica possible. This paper provides key insights into the improvement of SA production and the modelling of inhibition kinetics.

Techno-economic evaluation and life cycle assessment of a biorefinery using winery waste streams for the production of succinic acid and value-added co-products

This study presents techno-economic evaluation and life cycle assessment of a novel biorefinery using the three main waste streams generated by wineries for the production of bio-based succinic acid (SA), crude phenolic-rich extract, grape-seed oil, calcium tartrate and crude tannin-rich extract. Process design has been employed for the estimation of material and energy balances and the sizing of unit operations. The Minimum Selling Price of succinic acid production within a winery waste biorefinery ranges from $1.23–2.76/kgSA depending on the market price and the potential end-uses of the extracted fractions. The Global Warming Potential and the Abiotic Depletion Potential of winery waste valorisation through the proposed biorefinery are 1.47 kg CO2-eq per kg dry waste and 25.2 MJ per kg dry waste, respectively. Biorefining of winery waste could lead to the development of a sustainable and novel bioeconomy business model with new market opportunities and efficient waste management.

Restructuring the sunflower-based biodiesel industry into a circular bio-economy business model converting sunflower meal and crude glycerol into succinic acid and value-added co-products

A novel bio-economy business model for production of bio-based succinic acid and value-added co-products has been developed using biodiesel industry by-products. Sunflower meal (SFM) fractionation resulted in an antioxidant-rich extract via aqueous ethanol extraction and a protein concentrate stream via NaOH recycling using ultrafiltration. Solid state fermentation (SSF) by Aspergillus awamori using untreated SFM resulted in the production of cellulase, β-glucosidase, xylanase, protease and phytase. After the extraction of co-products, the residual SFM was hydrolysed via thermochemical and enzymatic treatment using crude enzymes resulting in conversion yields of 0.23 g g−1 total Kjeldahl nitrogen to free amino nitrogen, 0.56 g g−1 cellulose to glucose and 0.92 g g−1 hemicellulose to sugars. The SFM-derived hydrolysate was used in fed-batch cultures of Actinobacillus succinogenes leading to 34 g L−1 succinic acid with a yield of 0.6 g g−1 and a productivity of 0.71 g L−1 h−1. Crude glycerol supplemented with SFM hydrolysate resulted in 69.1 g L−1 succinic acid with a yield of 0.39 g g−1 and a productivity of 1.26 g L−1 h−1 by Yarrowia lipolytica PSA02004. SFM was efficiently used for co-product extraction, fermentation medium production and on-site crude enzyme production via SSF.

Optimization of fermentation medium for succinic acid production using Basfia succiniciproducens

The development of cost-competitive bioprocesses is dependent on the formulation of low cost and efficient fermentation medium. Thus, optimization of nutrient composition in fermentation media combining efficient metabolic product formation with cost-competitiveness is needed. Within this context, the main novelty of this study was to optimize the nutrient composition of a fermentation medium used for bio-based succinic acid production by the wild-type bacterial strain Basfia succiniciproducens JF4016 combining low cost with high succinic acid production efficiency. Two Plackett–Burman​ Designs, a central composite design and constraint optimization were sequentially implemented to estimate the optimum and cost-competitive fermentation medium composition. The two Plackett–Burman Designs identified NaH 2 PO 4 , Na 2 HPO 4 and NaCl as the most important medium components. Central composite design identified the optimum concentrations of NaH 2 PO 4 , Na 2 HPO 4 , NaCl, yeast extract and corn steep liquor leading to the highest productivity. Constraint optimization was employed to maximize productivity using constraints on the final succinic acid concentration, yield, by-products to succinic acid ratio and fermentation medium cost. Validation of the model predictions was carried out in a bioreactor culture at ca. 30 g/L initial glucose concentration. The optimum fermentation medium containing 19.4 g/L CSL, 9.2 g/L yeast extract, 3 g/L NaCl and 4.32 g/L sodium phosphates led to the production of 19 g/L succinic acid concentration, 0.62 g/g yield and 1.9 g/(L ⋅ h) productivity. • Experimental design to optimize fermentation medium for succinic acid production by Basfia succiniciproducens . • Succinic acid production reached 19 g/L with 0.62 g/g yield and 1.9 g/(L⋅h) productivity. • Reduced fermentation medium cost ($1.22/kg SA ) was estimated at the optimal medium composition.

Green hydrogen based succinic acid and biopolymer production in a biorefinery: Adding value to CO2 from acidogenic fermentation

Dark fermentative biohydrogen (bio-H2) production is usually associated with the formation of CO2 and organic acids as by-products. Although bio-H2 is an attractive low-carbon renewable fuel, generation of CO2 during production limits its use in certain applications. Nevertheless, these gases can be used for the production of various chemicals and materials, which are currently being manufactured from fossil carbon feedstocks. Therefore, in the present work we report an integrated approach for effective utilization of this bio-H2 and CO2 generated during acidogenic fermentation (AF) for the production of bio-based succinic acid (SA) using bacterial strain C. amalonaticus. In addition, the organic acids generated in the effluent after AF were converted into polyhydroxyalkanoates (PHA) using enriched mixed microbial consortia. Results show that continuous supply of CO2 positively affected SA production (14.7 g L-1) in comparison to control operation (12.07 g L-1) where CO2 was supplied at the beginning of the cycle. Bio-H2 acted as an effective electron donor for the formation of reduced end product, SA. The organic acids were effectively utilized and 23 gPHB/g DCW (dry cell weight) was achieved. Among the acids acetic acid was consumed in major proportion followed by butyric and valeric acids. The biopolymer obtained was a homopolymer of polyhydroxybutyrate (PHB), which was confirmed using FTIR analysis. The biopolymer also depicted good thermal properties, which was analyzed by TGA, DSC and DTA analysis. This work represents a successful attempt to develop an integrated system capable of simultaneously producing biobased SA and PHA, both, valuable bio-based products with a huge market potential.

Batch fermentation of succinic acid from cheese whey by Actinobacillus succinogenes under variant medium composition.

Bio-based succinic acid production has attracted global attention since its consideration as a potential replacement to petroleum-based platform chemicals. This study used three different CO2 sources, namely NaHCO3, K2CO3 and MgCO3 for fermentation of succinic acid (SA) by Actinobacillus succinogenes under three distinct substrate conditions i.e. lactose, whey and whey devoid of any supplements. Batch experiments were performed in both anaerobic flasks and 5L benchtop fermenter. SA fermentation in anaerobic flasks was unfettered by supplementary nutrients. However, fermentation in the benchtop fermenter devoid of supplementary nutrients resulted into 42% reduction in SA yield as well as lower SA productivities. Furthermore, a significant reduction of cell growth occurred in anerobic flasks at pH < 6.0, and complete termination of bacterial activity was noted at pH < 5.3. The highest SA titer, yield and productivity of 15.67g/L, 0.54g/g and 0.33g/L/h, respectively, was recorded from whey fermentation with MgCO3. The present study further highlights significant inhibitory effect of K2CO3 buffered medium on Actinobacillus succinogenes. Thus, we can claim that environmental pollution as well as costs of SA production from whey can be reduced by leveraging on whey residual nutrients to support the activity of Actinobacillus succinogenes.

Engineering of Yarrowia lipolytica transporters for high-efficient production of biobased succinic acid from glucose

BackgroundSuccinic acid (SA) is a crucial metabolic intermediate and platform chemical. Development of biobased processes to achieve sustainable SA production has attracted more and more attention in biotechnology industry. Yarrowia lipolytica has a strong tricarboxylic acid cycle and tolerates low pH conditions, thus making it a potential platform for SA production. However, its SA titers in glucose media remain low.ResultsIn this study, we screened mitochondrial carriers and C4-dicarboxylic acid transporters to enhance SA secretion in Y. lipolytica. PGC62-SYF-Mae strain with efficient growth and SA production was constructed by optimizing SA biosynthetic pathways and expressing the transporter SpMae1. In fed-batch fermentation, this strain produced 101.4 g/L SA with a productivity of 0.70 g/L/h and a yield of 0.37 g/g glucose, which is the highest SA titer achieved using yeast, with glucose as the sole carbon resource.ConclusionOur results indicated that transporter engineering is a powerful strategy to achieve the efficient secretion of SA in Y. lipolytica, which will promote the industrial production of bio-based SA.

Bioprocess development using organic biowaste and sustainability assessment of succinic acid production with engineered Yarrowia lipolytica strain

The utilization of crude waste streams and the development of novel bioprocessing routes are necessary in order to expand the industrial implementation of bio-based succinic acid production. This work focuses on the utilization of zero cost municipal organic biowaste for the production of bio-based succinic acid using the engineered yeast strain Yarrowia lipolytica PSA02004. Fermentation advances focused on pH regulation towards reduced NaOH requirements. Optimum initial carbon source concentration (100–120 g/L) in crude hydrolysates was estimated in shake flask cultures. Optimum kLα (183.2 h−1) in fed-batch cultures resulted in 42.2 g/L succinic acid with 0.38 g/g yield and 0.84 g/L/h productivity. A two-stage pH regulation strategy was employed to enhance succinic acid production efficiency and reduce NaOH requirements. The gradual reduction of pH from 6 to 5.5 resulted in 54.4 g/L succinic acid with 0.44 g/g yield and 0.82 g/L/h productivity and 43% lower NaOH consumption. The minimum selling price of succinic acid was estimated at $2.7/kg and $2.94/kg at annual production capacity of 50,000 t when the production cost of organic biowaste derived sugars were considered at $10/tTS and $110/tTS, respectively. The global warming potential and abiotic depletion potential were estimated at 2.72 kgCO2-eq/kgSA and 33.4 MJ/kgSA, respectively. Results showed that pH regulation is an important operating parameter towards the sustainable succinic acid production using municipal organic biowaste.

Valorisation of grape stalks and pomace for the production of bio-based succinic acid by Actinobacillus succinogenes

Valorisation of food supply chain waste for the production of bio-based platform chemicals will facilitate the transition to the sustainable bio-economy era. To this end, a novel process has been developed for the conversion of grape pomace and stalks into bio-based succinic acid. Free sugars (2.55 g/100 g), a phenolic-rich extract (185.75 mg gallic acid equivalent per g extract with Antioxidant Activity Index of 1.03) and lipids (8.2 g/ 100 g) were initially extracted from grape pomace. Residual grape pomace solids were subsequently mixed with grape stalks (1:1 ratio) and treated with sodium hydroxide (1%, w/v) for lignin removal. The residual solids were treated with varying H2SO4 concentrations (3–10 %, v/v) followed by enzymatic hydrolysis using different dosages of cellulases and β-glucosidase leading to the release of 22.7 g glucose per 100 g remaining solids (48 % cellulose hydrolysis). Actinobacillus succinogenes fed-batch cultures carried out with the crude hydrolysate as fermentation medium and free sugars from grape pomace as feeding solution resulted in 40.2 g/L succinic acid concentration with a yield of 0.67 g/g and productivity of 0.79 g/L/h. This study proposes for the first time a sustainable process for valorising grape pomace and stalks for efficient bio-based succinic acid production integrated with the extraction of value-added fractions.

Recent progress on bio-succinic acid production from lignocellulosic biomass.

Succinic acid is a valuable bulk chemical, which has been extensively applied in food, medicine, surfactants and biodegradable plastics industries. As a substitute for chemical raw material, bio-based succinic acid production has received increasing attention due to the depletion of fossil fuels and environmental issues. Meanwhile, the effective bioconversion of lignocellulosic biomass has always been a hot spot of interest owning to the advantages of low expense, abundance and renewability. Consolidated bioprocessing (CBP) is considered to be an alternative approach with outstanding potential, as CBP can not only improve the product yield and productivity, but also reduce the equipment and operating costs. In addition, the current emerging microbial co-cultivation systems provide strong competitiveness for lignocellulose utilization through CBP. This article comprehensively discusses different strategies for the bioconversion of lignocellulose to succinic acid. Based on the principles and technical concepts of CBP, this review focuses on the progress of succinic acid production under different CBP strategies (metabolic engineering based and microbial co-cultivation based). Moreover, the main challenges faced by CBP-based succinic acid fermentation are analyzed, and the future direction of CBP production is prospected.

Metabolic engineering of Aspergillus niger via ribonucleoprotein-based CRISPR\u2013Cas9 system for succinic acid production from renewable biomass

BackgroundSuccinic acid has great potential to be a new bio-based building block for deriving a number of value-added chemicals in industry. Bio-based succinic acid production from renewable biomass can provide a feasible approach to partially alleviate the dependence of global manufacturing on petroleum refinery. To improve the economics of biological processes, we attempted to explore possible solutions with a fungal cell platform. In this study, Aspergillus niger, a well-known industrial production organism for bio-based organic acids, was exploited for its potential for succinic acid production.ResultsWith a ribonucleoprotein (RNP)-based CRISPR–Cas9 system, consecutive genetic manipulations were realized in engineering of the citric acid-producing strain A. niger ATCC 1015. Two genes involved in production of two byproducts, gluconic acid and oxalic acid, were disrupted. In addition, an efficient C4-dicarboxylate transporter and a soluble NADH-dependent fumarate reductase were overexpressed. The resulting strain SAP-3 produced 17 g/L succinic acid while there was no succinic acid detected at a measurable level in the wild-type strain using a synthetic substrate. Furthermore, two cultivation parameters, temperature and pH, were investigated for their effects on succinic acid production. The highest amount of succinic acid was obtained at 35 °C after 3 days, and low culture pH had inhibitory effects on succinic acid production. Two types of renewable biomass were explored as substrates for succinic acid production. After 6 days, the SAP-3 strain was capable of producing 23 g/L and 9 g/L succinic acid from sugar beet molasses and wheat straw hydrolysate, respectively.ConclusionsIn this study, we have successfully applied the RNP-based CRISPR–Cas9 system in genetic engineering of A. niger and significantly improved the succinic acid production in the engineered strain. The studies on cultivation parameters revealed the impacts of pH and temperature on succinic acid production and the future challenges in strain development. The feasibility of using renewable biomass for succinic acid production by A. niger has been demonstrated with molasses and wheat straw hydrolysate.