Skip to main content
NCBI home page
3 Biotech logoLink to 3 Biotech
. 2025 Sep 15;15(10):348. doi: 10.1007/s13205-025-04522-z

Efficient bioconversion of deinked paper sludge into valuable biobased succinic acid by engineered Escherichia coli

Chotika Gosalawit 1, Walainud Congthai 1, Kanyarat Onsanoi 1, Chutchawan Phosriran 1, Kuan-Chen Cheng 2,3,4,5, Kata Buda 6, Csaba Feher 6, Kaemwich Jantama 1,✉
PMCID: PMC12436256  PMID: 40964658

Abstract

With the ongoing rise in petroleum prices, there has been growing interest in the microbial production of succinic acid from industrial wastes. Deinked paper sludge (DPS) is a significant waste, rich in cellulose and hemicellulose, generated in paper recycling plants. The disposal of DPS raises environmental concerns, and potential health hazards. This study explored an alternative and integrated platform, combining effective pre-treatment and enzymatic hydrolysis of DPS, and efficient fermentation process, for biobased succinic acid production by Escherichia coli KJ122. Initially, 10% (w/v) DPS was pre-treated with 1 N NaOH for 16 h followed by autoclaving at 120 °C for 20 min to remove inhibitors. The cellulase loading of 60 PCU (Protein Centered Unit)/g NaOH-pre-treated DPS was found to be optimal to release the highest fermentable sugars to 84.6 ± 1.8% after hydrolysis. For separated hydrolysis and fermentation (SHF), succinic acid at 27.80 ± 0.37 g/L with a yield of 0.68 ± 0.20 g/g and productivity of 0.50 ± 0.00 g/L/h was achieved. However, DPS loading at 100 g/L NaOH-pre-treated DPS in SHF posed challenges due to high viscosity of the broth, requiring increased agitation and energy consumption, and prolonged operating time, particularly for large-scale operations. To address these issues, a fed-batch semi-SHF process was employed, significantly improving succinic acid yield (0.97 ± 0.01 g/g; 88.1% theoretical maximum) and productivity (0.55 ± 0.02 g/L/h). According to the mass balance analysis, producing 1 kg of succinic acid required 2.5 kg NaOH-pre-treated DPS. This work provides an alternative and effective method for converting DPS into high-value bio-succinic acid, offering potential applications in the paper industry and contributing to bio-circular economy and zero-waste principles.

Keywords: Succinic acid, Deinked paper sludge, E. coli, Fermentation, Zero waste, Optimization

Introduction

Succinic acid is an organic acid recognized as one of the 12 valuable biobased chemicals by the United States Department of Energy (DOE) produced from lignocellulosic materials (Bozell and Petersen 2010). It is widely used as a building block for industrial productions of commodity and specialty chemicals and numerous everyday consumer products, pharmaceuticals, and adhesives, representing a total immediate addressable market in excess of $7.2 billion (Phosriran et al. 2024). To date, succinic acid is mostly produced through chemical processes using crude oil as a substrate due to economic competitiveness. However, various difficulties, such as the requirements of complex processes and extreme reaction conditions and high demand of energy consumption during synthesis, have been obstacles to commercially produce succinic acid (Phosriran and Jantama 2024).

A microbial fermentation by especially Escherichia coli strains has gained attraction in biobased succinic acid production. For decades, E. coli strains were metabolically engineered via gene deletion and overexpression of heterologous genes to enhance carbon fluxes through pathways of succinic acid production under both aerobic and anaerobic conditions. For example, Lee et al. (2005) showed that disruptions of glucose-specific phosphotransferase (PTS) transporter (ptsG) and pyruvate kinase (pykFA) genes enhanced succinic acid production more than sevenfold compared to the wildtype strain with the maximum yield of 0.35 mol/mol glucose (up to 2 g/L succinic acid) in E. coli W3110GFA. Huang et al. (2019) overexpressed carbonic anhydrase (ecaA) and phosphoenolpyruvate carboxylase (ppc) genes under the control of an anaerobically induced nitrate reductase (nirB) promotor derived from Salmonella enterica in E. coli HD134 possessing pflA, ldhA, and ptsG deletions. The strain increased succinic acid production up to 16.30 g/L with a yield of 0.54 mol/mol glucose. Chiang et al. (2021) also established E. coli BL-Suc by deleting succinate dehydrogenase (sdhA) and bacterial transcriptional regulator (arcA) genes to produce up to 8.59 g/L succinic acid with the yield of 0.28 mol/mol using glutamate and aspartate as substrates under aerobic conditions. However, most of developed fermentation processes depended upon requirements of pure substrates for producing succinic acid, leading to an increase in production costs regarding medium preparation when producing it on a large industrial scale. Alternatively, wastes from agricultural and agro-industrial sectors have gained attention as a feedstock for the fermentative production of succinic acid because of their abundant, environmentally friendly, and sustainable bioresource. There were then numerous lignocellulosic biomasses, such as Napier grass (Lee et al. 2022), sugarcane bagasse (Borges and Pereira 2011), wood hydrolysate (Zhu et al. 2020), orange peel (Akhtar et al. 2014), and potato and beer wastes (Escanciano et al. 2024) that were successfully utilized for succinic acid production by developed E. coli strains. These demonstrated and encouraged bioeconomy processes toward a concept of the zero-waste biorefinery and society.

Paper sludge (PS) is one of the huge wastes generated during papermaking and pulping processes. Presently, approximately 400 million wet tons in total annually of PS is generated, and Brazil, China, the United States, Canada, South Africa, and Thailand are majority contributors (Naicker et al. 2020). It is made in the form of short fibers containing organic matters > 60% (w/w) and its main components are cellulose, hemicellulose, lignin, and protein (Zhang et al. 2021). Deinked Paper Sludge (DPS) is one of the PS wastes generated in recycling paper plants. It also contains short cellulose fibers and a ton of manufactured recycling paper usually produces 150 kg of DPS (Bajpai 2024). General disposal methods of PS including DPS are landfilling, incineration, gasification, and pyrolysis. These raise in environmental concerns such as leachate contamination, greenhouse gas emissions, high energy consumption, and health hazards involving in disease epidemics due to the pathogen growth in PS (Pochyba et al. 2023). Previously, several efforts have been made to utilize PS to produce value-added biochemicals, such as hydrogen (Kádár et al. 2003), ethanol and isoprene (Duncan et al. 2020), and lactic acid (Budhavaram and Fan 2009) via fermentation processes. However, the optimal pre-treatment and enzymatic hydrolysis methods for converting PS into a high yield of fermentable sugars prior to fermentation have not been fully elucidated, presenting a key challenge in the efficient biorefinery utilization of PS. Furthermore, the valorization of PS for the sustainable production of high-value bio-succinic acid remains unexplored. Therefore, it is essential to develop an integrated biorefinery process that combines effective pre-treatment and enzymatic hydrolysis with a high-performance fermentation platform for succinic acid production. Such advancements could facilitate the industrial adoption of this technology in the near future.

Most previous studies on succinic acid production via lignocellulosic biomass fermentation have predominantly employed Actinobacillus succinogenes, a natural succinic acid-producing bacterium (Khunnonkwao et al. 2024). However, the cultivation of A. succinogenes requires complex media enriched with yeast extract and peptone to support its growth. Furthermore, strict anaerobic conditions, often achieved by supplementation with CO2 or H2 gases, are necessary to enhance succinic acid production. A high-density inoculum is first generated and subsequently transferred to the production medium to enhance yield and productivity of succinic acid. The use of complex nutrients and the requirement for high inoculum cell biomass contribute significantly to overall production costs, particularly in terms of media preparation and fermentation. Additionally, the presence of complex organic components in the medium causes complicated downstream processing, necessitating multiple purification steps and specialized equipment to achieve high succinic acid purity.

Alternatively, E. coli KJ122 has previously been shown to efficiently produce high concentrations and yields of succinic acid from pure glucose and xylose sugars (Khunnonkwao et al. 2024), and several lignocellulosic materials including cassava pulp (Sawisit et al. 2015a), sugarcane bagasse (Sawisit et al. 2015b), rice straw (Sawisit et al. 2018; Jampatesh et al. 2019), and waste office paper (Congthai et al. 2025). Unlike A. succinogenes, E. coli KJ122 efficiently produced succinic acid under simple anaerobic conditions without requirements of rich nutrients, and even CO2 and H2 gases. These may reduce costs dealing with medium preparation, purification and waste disposal. It is therefore of interest to explore an alternative fermentation process to valorize DPS into succinic acid using E. coli KJ122. In this study, a fermentation process suitable for the efficient production of succinic acid by E. coli KJ122 was investigated. The effects of crude cellulase and DPS loadings on succinic acid production were optimized, and the resulting fermentation processes were demonstrated to enhance the yield and productivity of succinic acid from DPS. The optimized process resulted in impressive succinic acid yield and average productivity by E. coli KJ122. This work not only highlights the potential of E. coli KJ122 for efficiently producing succinic acid but also presents an alternative approach for valorizing DPS, potentially reducing the environmental and health hazards associated with its disposal.

Materials and methods

Strain, medium, materials, and chemicals

Escherichia coli KJ122 was used for succinic acid production throughout this study (Jantama et al. 2008). Briefly, this strain was developed by deleting genes encoding lactate dehydrogenase (ldhA), alcohol dehydrogenase (adhE), phosphotransacetylase-acetate kinase (pta-ackA), pentameric formate channel with pyruvate formate lyase (focA-pflB), methylglyoxal synthase (mgsA), pyruvate oxidase (poxB), propionate kinase (tdcD), threonine decarboxylase (tdcE), aspartate aminotransferase (aspC), and the NAD-dependent malic enzyme (sfcA) without overexpressing heterologous genes (Fig. 1) followed by the metabolic evolution. After evolution, the glucose-specific PTS system was spontaneously inactivated while ATP-binding cassette (ABC) sugars transporters were de-repressed in E. coli KJ122, resulting in the simultaneous consumption of glucose, xylose, and arabinose without catabolite repression by the strain (Sawisit et al. 2015a). The evolved strain produced high succinic acid up to 82 g/L with yield and productivity of 1.51 mol/mol glucose (89% theoretical maximum) and 0.9 g/L/h, respectively. A VRE P3 crude cellulase cocktail with an activity of > 27,500 PCU (Protein Centered Unit)/g enzyme possessing combined activities of mainly cellobiohydrolase (exoglucanase) and endoglucanase, and trace xylanase, was purchased from Siam Victory Chemicals Co., Ltd., Thailand. According to the manufacturer’s instruction, one PCU (Protein Centered Unit) is defined as the amount of a combined enzymatic activity of exoglucanase and endoglucanase that releases 1 nmol of 4-methylumbelliferone from 4-methylumbelliferyl-β-d-galactoside per second (Congthai et al. 2025).

Fig. 1.

Fig. 1

Open in a new tab

Metabolic pathway of E. coli KJ122 to produce succinic acid. The red-yellow crosses represent the gene deletions. Gene and enzymes: pflB pyruvate formate lyase, adhE alcohol dehydrogenase, ldhA lactate dehydrogenase, ackA acetate kinase, tdcD propionate kinase, tdcE threonine decarboxylase E, aspC aspartate aminotransferase, aspA aspartase, ppc phosphoenolpyruvate carboxylase, pck phosphoenolpyruvate carboxykinase, poxB pyruvate oxidase, pdh pyruvate dehydrogenase complex, gltA citrate synthase, citDEF citrate lyase, mgsA methylglyoxal synthase, mdh malate dehydrogenase, fumABC fumarase isozymes, frdABCD fumarate reductase, and sfcA malic enzyme

(adapted from Jantama et al. 2008)

For preliminary experiments, Celluclast 1.5L (Sigma-Aldrich, Singapore) was used to release fermentable sugars from untreated DPS. Luria–Bertani (LB) medium was used for the maintenance, propagation, and seed preparation of E. coli KJ122. Alfredo Martinez version 1 (AM1) medium was used for succinic acid production. The medium was formulated and optimized according to the minimal requirement of nutrients to support growth and maintenance of E. coli (Khunnonkwao et al. 2024). The AM1 medium was composed of 1 mM Betaine-KCl, 1.5 mM MgSO4·7H2O, 2 mM KCl, phosphate ammonium salts (7.6 mM (NH4)2HPO4, 19.9 mM NH4H2PO4), and trace metal. Therefore, phosphate ammonium salts were the nitrogen source, while the carbon source was supplied into the AM1 medium via pure sugars or even fermentable sugars derived from lignocellulosic materials. The DPS used in the study was kindly provided from Hiang Seng Fibre Container Co., Ltd., Thailand.

Cell inoculum preparation

To prepare the cell inoculum, a single colony of E. coli KJ122 was transferred into a 250-mL shaking flask containing 50 mL of LB medium and incubated overnight at 37 °C with shaking at 200 rpm. The culture was then diluted to an initial OD550 of 0.1 and transferred to a 500-mL small-scale bioreactor containing 350 mL of AM1 medium supplemented with 50 g/L glucose and 100 mM KHCO3. The bioreactor was maintained at 37 °C with agitation at 200 rpm for 24 h before being used as the inoculum. The pH was kept at 7.0 using a 1:6 ratio of 6 M KOH and 3 M K2CO3.

Chemical pre-treatment processes of DPS

The chemical pre-treatment of DPS was adapted from Gurram et al. (2015), where 10% (w/v) DPS was soaked in either 1 N NaOH or 1 M HCl solution. The slurries were incubated at room temperature for 16 h. The mixtures were then autoclaved at 121 °C for 20 min and allowed to cool to room temperature. The NaOH- and HCl-pre-treated DPS were thoroughly washed with running water until the pH of the drained water reached 7.0. After washing, the pre-treated DPS samples were dried at 80 °C until a constant weight was achieved (with a moisture content of 5% w/w). The dried samples were stored in a dry place until further use.

Total sugar determination by acid hydrolysis

Complete acid hydrolysis was carried out following the method of Khunnonkawo et al. (2024). In this process, 2 g of untreated, NaOH-pre-treated, and HCl-pre-treated DPS were mixed with 10 mL of 72% (w/v) H2SO4 and subjected to vigorous agitation at 55 °C for 5 min. Deionized (DI) water was then added to the mixture to achieve a total volume of 200 mL. The mixture was autoclaved at 121 °C for 25 min. Afterward, the samples were centrifuged, and the supernatant was collected for the analysis of released sugars.

Enzymatic hydrolysis of untreated and NaOH-pre-treated DPS

Enzymatic hydrolysis was carried out in a 250-mL Erlenmeyer flask. Untreated and NaOH-pre-treated DPS at a concentration of 20 g/L were mixed with AM1 medium, and the mixtures were sterilized at 121 °C for 20 min. After sterilization, the slurries were cooled to 50 °C, and crude cellulase was added at loadings of 60, 80, or 100 PCU/g of pre-treated DPS, as indicated. The enzymatic hydrolysis reaction was conducted at 50 °C with agitation at 200 rpm for 96 h. During hydrolysis, 1 mL of the reaction mixture was aseptically collected at 12-h intervals. The collected samples were centrifuged at 15,000 rpm, and the supernatants were further analyzed for released fermentable sugars.

Succinic acid production from the NaOH-pre-treated DPS using fermentation processes

Succinic acid production from NaOH-pre-treated DPS was carried out using a 5 L Minifors 2 fermenter (Infors HT, Switzerland) with a total working volume of 2.5 L. The effect of initial solid content in the NaOH-pre-treated DPS (50, 75, and 100 g/L) on succinic acid production was first investigated with a crude cellulase loading of 60 PCU/g DPS. In the batch separate hydrolysis and fermentation (SHF) process, the NaOH-pre-treated DPS was sterilized by autoclaving at 121 °C for 20 min. Cellulase was then added to the slurry, and enzymatic hydrolysis was carried out at 50 °C with agitation at 400 rpm for 48 h. After hydrolysis, the temperature was reduced to 37 °C, and AM1 medium (Khunnonkwao et al. 2024) along with a 100 mM KHCO3 solution was added to the bioreactor to achieve a total working volume of 2.5 L. The inoculum, with an initial OD550 of 0.5, was introduced into the slurry mixture. The fermentation was conducted at 37 °C with agitation at 200 rpm for 50 g/L NaOH-pre-treated DPS and 400 rpm for 75–100 g/L NaOH-pre-treated DPS, without providing additional gases. The pH was maintained at 7.0 through automatic addition of the base solution.

In the batch simultaneous saccharification and fermentation (SSF) process, the NaOH-pre-treated DPS was sterilized by autoclaving at 121 °C for 20 min. The hydrolysis conditions were the same as those in the batch SHF process, except that enzymatic hydrolysis for pre-saccharification was performed for 6 h. Afterward, the temperature of the slurry was reduced to 37 °C, and the cell inoculum was added. The cultivation conditions were the same as those used in the batch SHF process. For the fed-batch semi-separate hydrolysis and fermentation (S-SHF) process, NaOH-pre-treated DPS at a concentration of 50 g/L was sterilized by autoclaving at 121 °C for 20 min. The temperature was then lowered to 50 °C, and cellulase along with AM1 medium was added to the fermenter, with an initial working volume of 2.5 L. The pre-saccharification by enzymatic hydrolysis was carried out at 50 °C with agitation at 400 rpm for 12 h. Afterward, the temperature was reduced to 37 °C before adding the inoculum. The cultivation was maintained at 37 °C with 200 rpm agitation.

To prepare the feeding reservoir, the hydrolysis of 60 g/L NaOH-pre-treated DPS was carried out in a total volume of 4 L at 50 °C and 400 rpm for 48 h. The slurry was then filtered through Whatman 4 filter paper (Cytiva, UK). The filtrate was sterilized at 121 °C for 20 min and centrifuged at 4000 rpm for 10 min. The supernatant was used as a liquid feeding sugar solution (14 g/L glucose + 5 g/L xylose) for intermittent feeding when the concentration in the fermentation broth during the fed-batch S-SHF process fell below 10 g/L. During fermentation, 2 mL of the fermentation broth was aseptically withdrawn from the vessel via the venting port at 8-h intervals. The samples were subsequently analyzed for cell density, and concentrations of fermentable sugars and metabolites.

Analytical method

Cell viable density was determined by serially diluting the broth samples, plating them onto LB agar, and incubating at 37 °C for 24–36 h. Cell viability was reported as CFU/mL. For the analysis of fermentation broth composition, the samples were centrifuged, and the supernatant was filtered through a 0.2-μm filter. The filtrate was then diluted with 4 mM H2SO4 at a 1:10 ratio. Sugars and organic acids in the diluted samples were quantified using HPLC (Agilent Technology 1200 series, Germany) with an Aminex® HPX-87H, 7.8 × 300 mm column (BioRad, USA) and a refractive index (RI) detector (RI-150, Thermo Spectra System, USA). The column temperature was set to 45 °C, and 4 mM H2SO4 was used as the mobile phase at a flow rate of 0.4 mL/min. The concentrations of sugars and organic acids were then used to calculate sugar consumption rates, concentrations, yields, and succinic acid productivity. In this study, glucose and xylose were only detected as fermentable sugars after performing acid and enzymatic hydrolyses.

Statistical analysis

Average values are presented with standard deviation (SD) based on three independent replicates (n = 3). To assess differences among the average values, analysis of variance (ANOVA) was conducted using GraphPad Prism 8.0.2 (GraphPad Software, USA). Tukey’s test was applied to determine significance at the 99% (p < 0.01) or 95% (p < 0.05) confidence level, as indicated.

Results and discussion

Pre-treatment of DPS by diluted NaOH and HCl solutions

The compositions of untreated, NaOH-pre-treated, and HCl-pre-treated DPS samples, determined by proximal analysis, are summarized in Table 1. The untreated DPS sample exhibited high cellulose and hemicellulose contents, totaling 89.85 ± 1.41% (w/w), and a low lignin content of 4.95 ± 0.47% (w/w). This observation is consistent with the findings of Rabemanolontsoa et al. (2024), who reported that recycled paper sludge contained only 3.1% (w/w) lignin, while cellulose and hemicellulose comprised up to 84.9% (w/w). Similarly, Quintana et al. (2024) revealed that raw waste sludge from the paper and pulp industry contained glucan derived from cellulose up to 88.6% (w/w), with no detectable lignin. These results indicate that paper sludge typically contains a low lignin content. Therefore, E. coli KJ122 was utilized to produce succinic acid from 100 g/L untreated DPS to elucidate whether the untreated DPS could be used directly for fermentation without performing the pre-treatment due to its low lignin content. Enzymatic hydrolysis of DPS using Celluclast 1.5L at enzyme loadings of 10 and 20 FPU/g DPS generated total fermentable sugars concentrations of 27.8 ± 2.5 g/L and 29.3 ± 1.1 g/L, respectively. These results indicate that a twofold increase in enzyme loading did not significantly enhance a sugar release from untreated DPS. Consequently, fermentation of the hydrolysates with E. coli KJ122 resulted in comparable succinic acid concentrations of 19.4 ± 0.3 g/L and 20.7 ± 1.0 g/L, respectively, with an overall yield of approximately 0.20 g/g untreated DPS in both cases. These results suggested that the resistance of untreated DPS to enzymatic hydrolysis at higher enzyme loading was not primarily due to enzyme shortage. However, it may be due to a poor cellulase accessibility to cellulose and hemicellulose fibers hindered by lignin or other factors affecting cellulase activity. Wang et al. (2021) indicated that toxic substances, such as vanillic acid, acetic acid, alcohol derivatives, furfural, and 5-hydroxymethylfurfural (5-HMF), which are produced from degraded lignin and sugars during papermaking processes, are commonly found in wastewater and residual compounds released from paper mills. These substances could potentially be transferred to DPS. However, these compounds were not detected in the untreated DPS sample before and after enzymatic hydrolysis as analyzed by HPLC. Therefore, it is hypothesized that the untreated DPS may contain other unidentified impurities that also affect the activity of Celluclast 1.5L. Gogoi et al. (2018) and Lopes et al. (2018) revealed that coating materials and chemicals used during paper production, including calcium carbonate, kaolin, titanium dioxide, and clay, are usually found in DPS after the deinking process. These compounds may contribute to the toxicity of DPS, inhibiting the activity of Celluclast 1.5L and negatively affecting microbial growth and succinic acid production. It is likely that the pre-treatment process is necessary to remove these toxic compounds in DPS, thereby improving the solubility and digestibility of the fibers, which would enhance the release of fermentable sugars during enzymatic hydrolysis.

Table 1.

Proximal analysis of untreated, HCl- and NaOH-pre-treated DPS samples

Samples¥ Cellulose
(%, w/w)		 Hemicellulose (%, w/w) Lignin
(%, w/w)		 Ash
(%, w/w)
Untreated DPS 82.23 ± 0.92α 7.62 ± 0.49α 4.98 ± 0.47α 5.42 ± 0.42α
NaOH-pre-treated DPS 88.65 ± 0.60β 2.40 ± 0.67β 2.72 ± 0.27β 6.23 ± 0.70α
HCl-pre-treated DPS 82.66 ± 3.86α 11.70 ± 3.64α 3.57 ± 0.81α 2.07 ± 0.10β
Open in a new tab

¥Data represented as average ± standard deviation (SD) from three independent experiments (n = 3)

Statistical analysis was calculated via an One-Way ANOVA. The different superscript Greek symbols indicate significant differences between mean values (p ≤ 0.01) in the same column

Following the pre-treatment of DPS with NaOH and HCl, the HCl-pre-treated DPS sample contained comparable levels of cellulose, hemicellulose, and lignin to those of the untreated DPS sample. In contrast, the NaOH-pre-treated DPS contained a significantly higher level of cellulose, but lower hemicellulose and lignin contents than those of untreated and the HCl-pre-treated DPS samples as determined by proximal analysis (Table 1). It is likely that the NaOH pre-treatment significantly altered the cellulose and hemicellulose compositions of DPS, and removed notable amounts of lignin and hemicellulose. Further, the complete acid hydrolysis by 72% (w/v) H2SO4 of 10 g/L untreated, NaOH-pre-treated, and HCl-pre-treated DPS samples was performed to investigate the effect of pre-treatments on levels of total fermentable sugars released from the pre-treated DPS. As shown in Table 2, the NaOH-pre-treated DPS provided the highest level of total fermentable sugars than those of the untreated and HCl-pre-treated DPS. Although the NaOH and HCl pre-treatments did not cause a significant release of total fermentable sugars from the pre-treated DPS samples, the glucose levels from the NaOH-pre-treated and HCl-pre-treated DPS samples were notably higher than that of the untreated DPS. In contrast, the xylose levels released from the HCl-pre-treated DPS were significantly lower compared to the untreated and NaOH-pre-treated DPS samples. This suggests that the HCl-pre-treatment effectively solubilized the hemicellulose fractions in DPS, whereas the NaOH pre-treatment did not. This may be due to the fact that hemicellulose is non-crystalline, and the branched structure makes it more reactive to acid than the tightly packed cellulose. Therefore, protons attack and hydrolyze glycosidic bonds between sugar units in hemicellulose into monomeric sugars (mostly xylose and arabinose), which are soluble in water as a black liquor (Jampatesh et al. 2019). However, both pre-treatments reduced the recalcitrance of the lignocellulosic fibers in DPS, as evidenced by a higher degree of hydrolysis by strong acid, reflected in the increased sugar release from the pre-treated DPS compared to the untreated DPS (Table 2).

Table 2.

Composition of sugars released from untreated, HCl- and NaOH-pre-treated DPS samples with different hydrolysis conditions

Samples¥ Glucose
(g/L)		 Xylose
(g/L)		 Total fermentable sugars (g/L) % Total released fermentable sugars
10 g/L DPS + 72% (w/v) H2SO4 (Strong acid digestion)
 Untreated 5.47 ± 0.24β 1.45 ± 0.21α 6.93 ± 0.43β 69.3 ± 4.3β
NaOH-pre-treated 6.93 ± 0.12α 1.53 ± 0.06α 8.46 ± 0.18α 84.6 ± 1.8α
 HCl-pre-treated 6.84 ± 0.12α 1.16 ± 0.01β 7.99 ± 0.12α 79.9 ± 1.2α
20 g/L Untreated DPS
 + 60 PCU/g 9.64 ± 0.23d 2.50 ± 0.08b 12.14 ± 0.24c 60.7 ± 0.1c
 + 80 PCU/g 9.66 ± 0.16d 2.54 ± 0.04b 12.20 ± 0.16c 61.0 ± 0.1c
 + 100 PCU/g 10.06 ± 0.11d 3.03 ± 0.13b 13.09 ± 0.24c 65.5 ± 0.1c
20 g/L NaOH-pre-treated DPS
 + 60 PCU/g 13.94 ± 0.38a 2.93 ± 0.47b 16.87 ± 0.72ab 84.4 ± 0.4ab
 + 80 PCU/g 12.77 ± 0.14c 3.58 ± 0.46ab 16.35 ± 0.55b 81.8 ± 0.3b
 + 100 PCU/g 13.40 ± 0.17b 4.45 ± 0.87a 17.85 ± 0.87a 89.2 ± 0.4a
Open in a new tab

¥Data represented as average ± standard deviation (SD) from three independent experiments (n = 3). The percentage of fermentable sugars released was calculated by dividing glucose and xylose as fermentable sugars released with total DPS provided

Statistical analysis was calculated via an One-Way ANOVA. The different superscript Greek symbols and lower-case letters indicate significant differences between mean values (p ≤ 0.01) in the same column for the acid hydrolysis and enzymatic hydrolysis respectively

Bayr et al. (2013) showed that applying strong acid pre-treatment at a high temperature up to 140 °C effectively solubilized the activated PS. However, this process also led to the formation of hexoses, pentoses, and lignin by-products, which were further degraded to produce inhibitors like furfural and 5-HMF. These inhibitors were harmful to anaerobic microorganisms, negatively affecting methane production. Alternatively, several studies have demonstrated that performing NaOH pre-treatment at milder temperatures and pressures caused swelling of the wood fiber cell wall. This significantly increased surface area, reduced crystallization, promoted the depolymerization of lignocelluloses, and disrupted lignin-carbohydrate bonds (Johannes and Xuan 2024; Kaur et al. 2019). Aghmashhadi et al. (2020) also revealed the NaOH pre-treatment combined with ultrasonics increased the released glucose concentration after enzymatic hydrolysis. Additionally, Gogoi et al. (2018) and Wang et al. (2021) showed that NaOH pre-treatment combined with low-temperature hydrothermal treatment of paper mill activated sludge could prevent the release of inhibitory compounds, improve sludge biodegradability, and enhance biogas productions in microbial electrolysis cells, as well as acetone–butanol–ethanol production, compared to acid pre-treatments. Based on these findings, the NaOH-pre-treated DPS, which exhibited the highest total released fermentable sugars (84.6%), was selected for further experiments on succinic acid production by E. coli KJ122. These results indicated the effectiveness of alkaline pre-treatment by NaOH in decreasing the cellulose crystallinity, and enhancing the enzyme accessibility by efficient removal of lignin, thereby increasing released fermentable sugars during enzymatic hydrolysis.

Alkaline pre-treatment by NaOH demonstrated superior efficacy in enhancing the enzymatic hydrolysis of lignocellulosic biomass compared to other pre-treatment methods (Show et al. 2023) due to higher removal of lignin and lower generation of inhibitors. However, it also generates substantial amounts of alkaline wastewater, commonly referred to as black liquor, as a by-product of biomass delignification (Bastos et al. 2021). The generation of this waste stream poses significant environmental concerns. In addition, large volumes of water are required to thoroughly wash the solid fraction of NaOH-pre-treated lignocellulosic biomass to neutralize its pH prior to enzymatic hydrolysis. This extensive washing step contributes significantly to the overall production cost (Wang et al. 2020). Consequently, recent research has focused on developing mitigation strategies, particularly the recycle and reuse of black liquor and wastewater streams. For example, Bastos et al. (2021) demonstrated the potential reuse of black liquor from NaOH pre-treatment of grass clippings to pre-treat newly incoming biomass streams, resulting in significant lignin removal capacity from 7.1 to 17.6% after five reuse cycles. Similarly, Tran et al. (2020) showed that reusing the black liquor from NaOH pre-treatment of rubber wood saw dust maintained an almost-equal efficiency, with an increased cellulose content from 41.2 to 52.8% compared between pre-treatments by fresh 2% (w/v) NaOH solution and reused black liquor stream. These findings suggest that black liquor recycling following NaOH pre-treatment of lignocellulosic biomass aligns with the development of a zero-discharge system and supports the principles of a circular waste economy. This sustainable approach also demonstrated economic advantages, particularly when cheap reagents such as NaOH were re-utilized.

The economic feasibility of biochemical conversion of lignocellulosic biomass remains constrained by the high costs associated with enzyme use (Tran et al. 2020). To address this limitation, various strategies have been explored to reduce cellulase costs, including protein engineering of cellulase enzymes, optimization of enzyme loading, supplementation with additives to enhance enzymatic activity, and recycling of cellulase by recovering enzymes from spent hydrolysate for reuse in subsequent hydrolysis cycles (Kim et al. 2019). Several techniques have demonstrated efficient cellulase recovery, such as enzyme immobilization, tannin-induced flocculation, desorption of inhibitors from cellulase using polyethylene glycol (PEG), and ultrafiltration with nano-membranes (Chen et al. 2013). Although these methods show promise, repeated enzyme recycling often results in a gradual loss of enzymatic activity. For example, Wang et al. (2016) reported a decrease in cellulase activity from 48.8 to 35.1% after five hydrolysis cycles. Similarly, Kim et al. (2019) observed a decline in glucose yield from 96% to below 5% after the third reuse cycle of cellulase, primarily due to product inhibition by glucose, enzyme denaturation, and non-specific binding of cellulase to insoluble substrates. Nonetheless, such limitations can be mitigated by the addition of PEG, which reduces non-productive binding and facilitates the removal of glucose from the hydrolysate (Kim et al. 2019). Together, the integration of alkaline recycling and cellulase recovery presents a sustainable and economically viable approach for lignocellulosic bioconversion, contributing to improved chemical utilization and waste management in not only succinic acid production by E. coli KJ122 but also other biobased biochemical productions.

Effect of cellulase loadings on fermentable sugars released from the NaOH-pre-treated DPS

Commercially pure cellulases typically consist of various enzyme classes, including endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases activities (Lopes et al. 2018) which break down cellulosic fractions into monomeric fermentable sugars found in lignocellulosic materials. Several studies have demonstrated that increasing levels of a commercial Cellic CTec2 cellulase positively influenced the release of fermentable sugars from lignocellulosic materials. For example, Olszewska-Widdrat et al. (2023) demonstrated that applying cellulase at dosages ranging from 2.6 to 13.2 FPU/g sulfite fiber sludge significantly increased glucose concentrations from 44.0 to 94.1 g/L. Duncan et al. (2020) reported that increasing cellulase loadings to 2.3, 5.7, and 9.1 FPU/g paper mill sludge enhanced the level of glucose released from the sludge, achieving maximum glucose yields of 3.1%, 5.7%, and 14.2%, respectively. Additionally, Budhavaram and Fan (2009) observed that while cellulose conversion rates of paper sludge did not differ markedly between enzyme loadings of 10 and 20 FPU/g, d-(−)-lactic acid production by Bacillus coagulans from the PS hydrolysate was notably higher at 20 FPU/g under semi-continuous simultaneous saccharification and co-fermentation conditions. These findings underscore the importance of optimizing enzyme dosage to balance substrate conversion efficiency with overall product yield in lignocellulosic bioconversion processes. However, these studies appeared to require high concentrations and purity of the cellulase to fully hydrolyze cellulose and hemicellulose fibers in paper sludges. These raise concerns about the economic feasibility of bioproduct production from paper sludges due to the high cost of enzyme and the time needed for hydrolysis (Reyes-Sosa et al. 2017; Fennila and Shastri 2016). Therefore, it is essential to identify alternative, cost-effective sources of cellulase enzymes that exhibit catalytic performance comparable to that of the commercial Cellic CTec2.

In this study, an industrial-grade crude cellulase cocktail was used for the enzymatic hydrolysis of DPS, rather than commercially pure cellulases, in order to reduce production costs. The impact of crude cellulase loading on the release of fermentable sugars during enzymatic saccharification of DPS was investigated. During enzymatic hydrolysis of both untreated and NaOH-pre-treated DPS samples, glucose, xylose, and total fermentable sugar were rapidly released within the first 12 h of incubation, followed by a more gradual increase in the sugar release throughout the remaining incubation period (Fig. 2). For untreated DPS, the release rates of glucose and xylose were relatively comparable, regardless of crude cellulase loadings ranging from 60 to 100 PCU/g (Fig. 2A–C). After 96 h of incubation, no statistically significant differences were detected in the concentrations of glucose, xylose, or total fermentable sugars at any enzyme loading (Table 2). This lack of response to increased cellulase dosage suggests that the intrinsic recalcitrance of untreated DPS was primarily due to its high lignin content (Table 1) that restricted cellulase accessibility to lignocellulosic fibers, thereby limiting the extent of hydrolysis. In contrast, NaOH-pre-treated DPS provided relatively higher sugar releasing rates during the first 12 h (Fig. 2D–F) and yielded significantly higher concentrations of glucose, xylose, and total fermentable sugars than untreated DPS after 96 h of enzymatic hydrolysis (Table 2). At 60 PCU/g cellulase loading, NaOH-pre-treated DPS produced 13.94 ± 0.38 g/L glucose, 2.93 ± 0.47 g/L xylose, and 16.87 ± 0.72 g/L total fermentable sugars, exceeding the corresponding values for untreated DPS. The superior performance can be attributed to the approximately twofold reduction in lignin content achieved by NaOH pre-treatment (Table 1), which increased cellulose accessibility and facilitated sugar release. However, further increases in cellulase loading beyond 60 PCU/g (to 80 or 100 PCU/g) did not significantly enhance total sugar release from NaOH-pre-treated DPS (Table 2). Considering the glucose release, the highest xylose and total fermentable sugar concentrations reached 4.45 ± 0.87 g/L and 17.85 ± 0.87 g/L at 100 PCU/g, respectively, whereas the highest glucose concentration (13.94 ± 0.38 g/L) was observed at 60 PCU/g. The lower glucose yields at higher enzyme loadings are likely due to feedback inhibition of endoglucanase and β-glucosidase activities by accumulated cellobiose and glucose during prolonged hydrolysis, as previously reported (Sawisit et al. 2015a; Jampatesh et al. 2019; Khunnonkwao et al. 2024).

Fig. 2.

Fig. 2

Open in a new tab

Enzymatic hydrolysis of 20 g/L untreated DPS with crude cellulase loadings of 60 PCU/g (A), 80 PCU/g (B), and 100 PCU/g (C). Enzymatic hydrolysis of 20 g/L NaOH-pre-treated DPS with crude cellulase loadings of 60 PCU/g (D), 80 PCU/g (E), and 100 PCU/g (F). The vertical bars represent standard deviations (n = 3). Glucose (triangle), xylose (circle), and total sugar (square)

As crude cellulase loadings increased, the concentration of released xylose improved, while glucose levels were highest at 60 PCU/g NaOH-pre-treated DPS and did not show significant differences. This can be attributed to the fact that the crude cellulase cocktail used in this study contained at least endoglucanase, β-glucosidase, and cellobiohydrolase, with a small amount of xylanase (Jampatesh et al. 2019). The glucose released during enzymatic hydrolysis was a key factor inhibiting the activity of both endoglucanase and β-glucosidase, leading to the accumulation of cellobiose, which in turn further inhibited cellulase activity (Xiao et al. 2004). In contrast to glucose, five-carbon sugars like xylose did not interfere with β-glucosidase or xylanase activities but may have slightly affected cellulose hydrolysis by endoglucanase (Zhai et al. 2018). For these reasons, no improvement in the glucose release was observed, while xylose levels increased as crude cellulase loadings were raised. Last, the total released fermentable sugar from enzymatic hydrolysis of the untreated DPS (60.7–65.5%) were lower than that of complete acid hydrolysis (69.3%), while those for the NaOH-pre-treated DPS showed a significant improvement (81.8–89.2%) (Table 2). An enzymatic loading of 60 PCU/g was chosen for subsequent saccharification experiments to evaluate succinic acid production via fermentation processes. To conclude, the data clearly demonstrated that NaOH pre-treatment resulted in the most effective lignin removal from DPS, as evidenced by the lowest lignin content and the highest levels of cellulose and hemicellulose in the NaOH-pre-treated DPS, compared to untreated and HCl-pre-treated DPS samples. Furthermore, results from enzymatic digestion and strong acid hydrolysis revealed that NaOH-pre-treated DPS exhibited the greatest susceptibility to enzymatic degradation and acid hydrolysis, indicating an enhanced substrate accessibility. These findings provide strong evidence that NaOH pre-treatment significantly improves the digestibility of DPS, highlighting its potential for large-scale application in the preparation of lignocellulosic substrates for succinic acid production.

Succinic acid production from the NaOH-pre-treated DPS by SHF process

Succinic acid production by E. coli KJ122 was initially examined under the SHF process using different concentrations of the NaOH-pre-treated DPS at 50 g/L, 75 g/L, and 100 g/L. As shown in Fig. 3A–C, the initial glucose concentrations released from the NaOH-pre-treated DPS were rapidly consumed within 40 h of fermentation, while xylose was gradually consumed. This suggests that E. coli KJ122 co-utilized both glucose and xylose, despite the fact that glucose is typically preferred over xylose by microorganisms. This may be explained that the mgsA gene was deleted in E. coli KJ122, likely contributing to improved mixed sugar consumption. The methylglyoxal synthase, encoded by mgsA gene, catalyzes the conversion of dihydroxyacetone phosphate (DHAP), a glycolytic intermediate, to methylglyoxal, particularly under high carbon flux conditions, such as elevated glucose concentrations. As methylglyoxal is toxic, the accumulation of methylglyoxal usually inhibits cell growth and sugar consumptions. To relieve its detrimental effects, E. coli further metabolizes methylglyoxal by the glyoxalase enzymes to d-(−)-lactic acid, serving as a cellular detoxification mechanism (Fig. 1). Disruption of genes involved in the methylglyoxal biosynthesis pathway, including the methylglyoxal synthase (mgsA) gene, enhanced the co-utilization of sugars like xylose alongside glucose, improved sugar consumption, and increased succinic acid production (Jantama et al. 2008). Succinic acid was detected within the first 8 h of incubation and was produced in large quantities until glucose and xylose were exhausted, with a slight increase continuing until the end of fermentation at 72 h. The 50 g/L NaOH-pre-treated DPS resulted in the highest yield at 0.75 g/g sugars consumed and 0.36 g/g NaOH-pre-treated DPS provided. The highest succinic acid concentration and productivity were achieved at 27.80 g/L and 0.50 g/L/h, respectively, with a yield of 0.68 g/g sugars consumed when 100 g/L NaOH-pre-treated DPS was used. However, the yield and gross yield of succinic acid were not significantly different between 75 and 100 g/L NaOH-pre-treated DPS (Table 3). Even though the growth of E. coli KJ122 significantly decreased (Fig. 3C), the highest succinic acid production was likely due to the highest initial sugar content, which enabled the strain to utilize sugars at the fastest consumption rate.

Fig. 3.

Fig. 3

Open in a new tab

Succinic acid production by SHF process using the NaOH-pre-treated DPS of 50 g/L (A), 75 g/L (B), and 100 g/L (C). The vertical bars represent standard deviations (n = 3)

Table 3.

Fermentation profile of succinic acid production from SHF, SSF, and S-SHF processes using E. coli KJ122 with the NaOH-pre-treated DPS

Mode NaOH-pre-treated DPS
(g/L)		 Total fermentation time (h) Max. cell viability
(1011 CFU/mL)		 Total sugars consumption rate
(g/L/h)		 Succinic acidα By-products
Concentration (g/L) Yieldβ
(g/g)		 Gross yield
(g/g)		 Average productivity (g/L/h) Acetate (g/L) Lactate (g/L)
SHF 50 72 10.08 ± 2.64 0.64 ± 0.01d 17.96 ± 1.81bc 0.75 ± 0.06abc 0.36 ± 0.04ab 0.32 ± 0.02ab 4.47 ± 0.71bc 3.50 ± 0.06bc
75 72 4.10 ± 0.70 0.84 ± 0.00ab 22.71 ± 1.86b 0.62 ± 0.05c 0.30 ± 0.02b 0.41 ± 0.03ab 6.68 ± 0.01a 3.47 ± 0.54c
100 72 0.34 ± 0.16 0.94 ± 0.01a 27.80 ± 0.37a 0.68 ± 0.02bc 0.28 ± 0.00b 0.50 ± 0.00ab 5.94 ± 0.38ab 5.94 ± 1.06b
SSF 50 72 3.40 ± 0.10 0.24 ± 0.00e 15.42 ± 2.73c 0.89 ± 0.17ab 0.31 ± 0.06ab 0.28 ± 0.05ab 4.34 ± 0.48c 2.98 ± 0.36c
75 72 3.00 ± 0.60 0.31 ± 0.01de 13.77 ± 0.12c 0.59 ± 0.02c 0.17 ± 0.00c 0.24 ± 0.00ab 5.52 ± 0.26abc 5.59 ± 0.88b
100 72 1.75 ± 0.25 0.38 ± 0.01 cd 7.91 ± 0.09d 0.30 ± 0.01d 0.08 ± 0.00d 0.14 ± 0.00b 6.04 ± 0.26ab 9.18 ± 1.11a
Fed-batch S-SHF 50 48 7.00 ± 0.23 0.47 ± 0.01bc 21.82 ± 0.76b 0.97 ± 0.01a 0.40 ± 0.01a 0.55 ± 0.02a 4.26 ± 0.53c ND
Open in a new tab

SHF separate hydrolysis and fermentation, SSF simultaneous saccharification and fermentation, S-SHF semi-SHF, ND not detected

αData represented as average ± standard deviation (SD) from three independent experiments (n = 3). Different superscript letters indicate significant differences between mean values (p ≤ 0.05) in the same column

βYield is expressed as g succinic acid produced per g total fermentable sugars consumed. Gross yield is expressed as g succinic acid produced per g NaOH-pre-treated DPS used. Average productivity is calculated when the sugar utilization was exhausted

It was observed that the biosynthesis of cell biomass for E. coli KJ122 was relatively low during succinic acid fermentation. Jantama et al. (2008) explained that E. coli KJ122 was metabolically engineered to enhance succinic acid production under anaerobic conditions by directing carbon flux through the reductive arm of the TCA cycle (Fig. 1). This was achieved by deleting genes responsible for by-product formation, including lactate (ldhA), formate (pflB), ethanol (adhE), and acetate (ackA and tdcD). Notably, deletion of ackA and tdcD impaired ATP generation via substrate-level phosphorylation, limiting ATP yield to 1 mol per mol glucose during glycolysis, which restricts biomass biosynthesis. Under high DPS loadings, the strain likely redirected ATP usage toward cellular maintenance rather than growth, due to increased osmotic stress, as evidenced by reduced cell viability (Fig. 3C). To compensate for limited ATP, E. coli KJ122 further increased carbon flux through the reductive TCA pathway. One mol of ATP was additionally generated via phosphoenolpyruvate carboxykinase (pckA), while NADH was re-oxidized through malate and fumarate reduction by malate dehydrogenase and fumarate reductase, respectively. This metabolic shift accelerated sugar utilization and succinic acid production at high initial sugar concentrations. Similar phenomena were observed by Jampatesh et al. (2019) and Khunnonkwao et al. (2024) using high solid contents of rice straw and empty fruit palm bunches as substrates for succinic acid production with E. coli KJ122.

As the DPS concentration increased, the viscosity of the fermentation broth also rose, particularly at 100 g/L NaOH-pre-treated DPS. This had negative effects on the growth of E. coli KJ122, as evidenced by a sharp decline in maximum cell viability (Fig. 3C). These results suggested that at higher NaOH-pre-treated DPS concentrations, mass and heat transfer became less effective. It is likely that a higher agitation speed and power input was required for efficient mixing, but such conditions may not be ideal for large-scale succinic acid production. Therefore, the use of 50 g/L NaOH-pre-treated DPS, which provided the highest succinic acid yield and gross yield with a balanced concentration and productivity, appears to be more suitable for succinic acid production than using higher DPS concentrations. To mitigate the negative effects of high-viscosity slurries during fermentation, many previous studies focusing on bioproduct production from paper sludges have used sugar solutions derived from enzymatic hydrolysates. Olszewska-Widdrat et al. (2023) demonstrated succinic acid production by Actinobacillus succinogenes B1 and Basfia succiniciproducens B2 strains using sulfite fiber sludge (SFS) hydrolysate supplemented with yeast extract and peptone. In their study, high SFS loading (150 g/L) was enzymatically digested at the pilot scale (50 L), and the resulting slurry was filtered and concentrated. Similarly, Duncan et al. (2020) prepared a sugar solution from 150 g/L paper mill sludge (PMS) solids by digesting with Cellic Ctec2 cellulase. This sugar solution, containing 50 g/L total sugars supplemented with yeast extract, was used in the SHF process to avoid the high viscosity of the fermentation broth caused by high PMS loadings during isoprene and ethanol production by E. coli and an industrial yeast strain. However, low yields of isoprene and ethanol were observed due to the presence of impurities and inhibitors in the hydrolysate. In contrast, directly utilizing DPS in this study can simplify the fermentation process by eliminating multiple complex steps, while still maintaining high succinic acid production efficiency, both in terms of yield and productivity, by E. coli KJ122. Moreover, using the mineral salts AM1 medium (containing 4 g/L of salts in total) without the addition of rich, complex nutrients (yeast extract and peptone) in our study may help reduce the cost of succinic acid production, particularly in terms of medium preparation and waste disposal.

Succinic acid production from the NaOH-pre-treated DPS by SSF process

In this study, the SSF process was employed with the aim of reducing the fermentation time for succinic acid production. The NaOH-pre-treated DPS was pre-digested for 6 h before the inoculum was added to the bioreactor. As shown in Fig. 4, the initial glucose and xylose produced during the pre-saccharification step were completely consumed within 24 h when 50 g/L and 75 g/L NaOH-pre-treated DPS were used (Fig. 4A, B), while these sugars were nearly depleted after 40 h in the 100 g/L NaOH-pre-treated DPS (Fig. 4C). The SSF process with 50 g/L NaOH-pre-treated DPS achieved the highest succinic acid concentration of 15.42 ± 2.73 g/L, comparing to those of 75 and 100 g/L NaOH-pre-treated DPS concentrations (Table 3). Notably, succinic acid production was rapid until the sugars were almost exhausted, after which its concentration reached maximum, even when fermentation was prolonged to 72 h. During SSF, E. coli KJ122 exhibited the highest concentration, yield, and productivity when utilizing 50 g/L NaOH-pre-treated DPS with low levels of by-products like acetate and lactate. This suggested that nearly all of the carbon flux derived from the released fermentable sugars was directed toward succinic acid production. In contrast, except for the 100 g/L NaOH-pre-treated DPS, succinic acid was not the major product in the fermentation process; most of the carbon flux was redirected toward acetate and lactate formation. Compared to the SHF process, the succinic acid production efficiency was significantly reduced during the SSF process at higher NaOH-pre-treated DPS concentrations. This indicates that the release of fermentable sugars from the NaOH-pre-treated DPS was insufficient to simultaneously convert the released sugars into succinic acid. It was likely that the 6-h pre-saccharification step prior to SSF was not optimal when high DPS loadings was provided. This negative effect was likely compounded by the high DPS loadings, which increased the viscosity and osmotic pressure of the medium, ultimately hindering mass and heat transfer, and thus affecting the availability of carbon substrates and nutrients for cellular metabolism, which in turn impacted succinic acid production by E. coli KJ122. These findings are consistent with the study by Takano and Hoshino (2016), which showed that increasing the initial PMS concentration from 50 to 100 g/L decreased both the concentration and yield of lactic acid produced by Rhizopus sp. However, these issues could be mitigated by extending the pre-saccharification step of the pre-treated biomass before initiating the SSF process (Gosalawit et al. 2024).

Fig. 4.

Fig. 4

Open in a new tab

Succinic acid production by SSF process using the NaOH-pre-treated DPS at the concentrations of 50 (A), 75 (B), and 100 g/L (C). The vertical bars represent standard deviations (n = 3)

SSF process has been widely applied for biochemical productions to reduce incubation time and operating costs. However, one major drawback is the required operating temperature, which is typically 50 °C for optimal cellulase activity, while microbial growth is best at a lower temperature of around 37 °C. This temperature mismatch leads to a reduced rate of enzymatic hydrolysis, resulting in a sugar limitation reflected in slower sugar consumption rates, which negatively impacts succinic acid production during SSF (Fig. 3, Table 3). Additionally, DPS may contain toxic compounds from the repulping and recycling processes, and further inhibitors are generated during pre-treatment. It is interesting to note that inhibitors derived from degraded lignin and sugars during NaOH pre-treatment, including vanillic acid, formic acid, furfural, and 5-HMF, were not detected in the enzymatic reactions and even fermentation broth containing the NaOH-pre-treated DPS in this experiment as analyzed by HPLC. Jampatesh et al. (2019) revealed that E. coli KJ122 tolerated up to 3.0 g/L acetic acid, 1.1 g/L formic acid, 0.5 g/L furfural, and 1.6 g/L 5-HMF. However, the accumulation of these inhibitors may reach a level of effective toxicity, but not yet at the minimum inhibitory concentration, especially at higher DPS concentrations during SSF process, negatively impacting enzymatic hydrolysis efficiency, the growth of E. coli KJ122, and succinic acid production. Our findings then suggest that the SSF process may not be ideal for succinic acid production from DPS using E. coli KJ122.

Succinic acid production by fed-batch semi-SHF from the NaOH-pre-treated DPS

To further reduce the operating time of succinic acid production using the SHF process, a fed-batch semi-saccharification SHF (S-SHF) approach was implemented. In this method, pre-saccharification was carried out for up to 12 h, after which the hydrolysis was stopped by autoclaving before inoculation. As illustrated in Fig. 5, glucose was consumed, and its concentration dropped to below 10 g/L at 16 h of incubation when the sugar solution derived from the pre-hydrolyzed DPS was introduced. At 48 h, succinic acid reached 21.82 ± 0.76 g/L with the yields of 0.97 ± 0.01 g/g sugar consumed and 0.40 ± 0.01 g/g NaOH-pre-treated DPS provided, and a productivity of 0.55 ± 0.02 g/L/h (Table 3). Maximum cell viability was observed at 40 h of fermentation. As shown in Table 3, the fed-batch S-SHF process produced succinic acid at a comparable concentration but significantly improved yields, productivity, and maximum cell viability compared to the SHF and SSF processes. This was due to the fact that the pre-saccharification helped strengthening shear forces during mixing that accelerated the release of fermentable sugars that supported the microbial growth. The succinic acid yield (0.97 g/g) achieved by the fed-batch S-SHF process was 88.2% of the theoretical maximum succinic acid yield (1.1 g/g sugars consumed) reported by Phosriran and Jantama (2024). Furthermore, the total operating time for the S-SHF process was reduced to 54 h (6 h for pre-saccharification and 48 h for fermentation), while the SHF process required 120 h in total (48 h for enzymatic hydrolysis and 72 h for fermentation). Our results align with Gosalawit et al. (2024), who demonstrated that the pre-saccharification step prior to a fed-batch S-SSF process using Klebsiella oxytoca KIS004-91T significantly enhanced d-(−)-lactic acid production from sugarcane bagasse. Compared to batch SHF (72 h for enzymatic hydrolysis and 48 h for fermentation), the fed-batch S-SSF process (12 h for pre-saccharification and 28 h for fermentation) improved lactate production without affecting d-(−)-lactate yield. Additionally, when compared to the batch SSF (4 h for pre-saccharification and 48 h for fermentation), the fed-batch S-SSF approach increased d-(−)-lactate concentration, yield, and productivity by 172%, 59.7%, and 231.1%, respectively. These results clearly confirmed that the pre-saccharification strategy of lignocellulose materials prior to fermentation helped not only reducing fermentation time but also increasing fermentation performances.

Fig. 5.

Fig. 5

Open in a new tab

Fermentation profile of fed-batch semi-SHF process using E. coli KJ122 with 50 g/L initial NaOH-pre-treated DPS. The vertical bars represent standard deviations (n = 3)

Surprisingly, no lactic acid was detected during the fed-batch S-SHF process, whereas it was observed in the SHF and SSF processes after 24 h of incubation (Table 3), despite the deletion of genes encoding enzymes responsible for lactate production, d-lactate dehydrogenase (ldhA) and methylglyoxal synthase (mgsA), in E. coli KJ122. This may be attributed to lactic acid production being activated by a membrane-associated l-lactate dehydrogenase, encoded by the lldD gene (Lopez-Campistrous et al. 2005). During the SHF and SSF processes, the high viscosity of the fermentation broth likely hindered mass and heat transfer between the fermentation broth and the cells. This non-homogeneous mixing likely disturbed the distribution of carbon substrates between the cells and the broth, potentially causing fluctuations in intracellular carbon fluxes and redox balance (NADH/NAD+ ratio) during metabolism. As a result, E. coli KJ122 may have diverted accumulated pyruvate from glycolysis through the lactate-producing pathway via l-lactate dehydrogenase activity, which operates at a faster rate than the succinic acid-producing pathway, to mitigate the detrimental effects of metabolic imbalances. This was evident as lactate production increased with higher DPS loadings, especially during the SSF process with 100 g/L NaOH-pre-treated DPS. In contrast, the fed-batch S-SHF process achieved more homogeneous mixing, as the feeding solution diluted the solid content in the fermentation broth, improving mass and heat transfer efficiency. This helped stabilizing the redox and metabolic conditions, allowing E. coli KJ122 to produce succinic acid without diverting carbon flux to lactate production.

Succinic acid produced via microbial fermentation has emerged as a promising sustainable alternative to petrochemically derived maleic anhydride (Orjuela et al. 2013). However, the fermentation broth typically contains a complex mixture of cells, residual substrates, proteins, and various by-products, including organic acids. These components complicate downstream processing, making efficient purification essential to achieve high yield and purity of succinic acid (Sadare et al. 2021). A range of purification strategies have been investigated, including solvent extraction, adsorption, esterification, electrodialysis, microfiltration, nanofiltration, and crystallization (Loffredo et al. 2024; Sadare et al. 2021). For instance, Lee et al. (2022) achieved a succinic acid purity of 99% and a recovery yield of 74% from the fermentation broth of A. succinogenes cultivated with Napier grass hydrolysate, using ultrafiltration coupled with single-stage electrodialysis. Jokodola et al. (2022) reported a recovery of over 75% succinic acid from xylose-based fermentation using vacuum concentration and direct crystallization following acidification. Using the fermentation broth of E. coli KJ122, Umpuch et al. (2016) achieved the succinic acid purity of 99.1% and the recovery yield of 30.25% via reactive extraction by 0.25 mol Tri-n-octylamine/kg 1-octanol under pH 2.4, followed by crystallization of the distilled broth at 4 °C. Additionally, Khunnonkwao et al. (2018) demonstrated that a purification process integrating nanofiltration with subsequent reverse osmosis achieved the succinic acid purity of 99.5% and the recovery yield of 92% from the fermentation broth. Even though downstream processing was not evaluated in the present study, E. coli KJ122 exhibited efficient succinic acid production from the NaOH-pre-treated DPS using the cost-effective, low-nutrient AM1 medium, with minimal formation of by-products. When combined with established purification strategies, it underscores the potential of E. coli KJ122 as a robust biocatalyst for the industrial-scale succinic acid production from renewable lignocellulosic feedstocks. The integrated approach yet offers a promising and sustainable platform for manufacturing biobased succinic acid.

A mass balance analysis for succinic acid production from DPS has been proposed (Fig. 6) based on the result obtained from the fed-batch S-SHF process. Approximately 1 kg of NaOH-pre-treated DPS is needed to produce 400 g of succinic acid. Therefore, to produce 1 kg of succinic acid, about 2.5 kg of NaOH-pre-treated DPS is required. To approximately estimate the production cost for achieving 1 kg succinic acid, costs regarding to medium preparation and substrate preparation in fermentation process have been only considered due to limited information about energy consumption, and purification and separation. Based on data from Table 3 and Fig. 6, the NaOH-pre-treated DPS about 55 g was totally provided into 1 L of AM1 medium. Since 2.5 kg of NaOH-pre-treated DPS has been proposed to produce 1 kg succinic acid, approximate 45.5 L of AM1 medium is then needed. According to Chan et al. (2016), the cost of AM1 medium was estimated to be about $0.424 USD/L. The total cost of AM1 medium required to produce 1 kg succinic acid is $19.292 USD. To obtain 2.5 kg NaOH-pre-treated DPS, 1.05 kg NaOH, which is sold at the price of $10 USD/kg (Atelier Evia Co., Ltd, Quebec, Canada), is required to pre-treat 2.63 kg DPS. To hydrolyze 2.5 kg NaOH-pre-treated DPS, total crude cellulase of 150,000 PCU is also mandatory for enzymatic hydrolysis. According to the manufacturer’s price, 1 g of crude cellulase, containing 27,500 PCU, costs $0.011 USD. The total cost of crude cellulase used is about $0.0583 USD. Therefore, the subtotal cost dealing with the medium preparation and DPS hydrolysis has been proposed to be $29.302 USD to produce 1 kg succinic acid. However, it is worth to note that costs dealing with utilities, energy inputs, and purification and separation about 10–20% of the medium preparation cost are recommended to be added (Werpy and Petersen 2004). Finally, the total production cost has been proposed between $32.232 and $35.162 USD per 1 kg succinic acid produced. Considering the market price of succinic acid, it is sold in the range of $60–$250 USD/kg, depending on purity and vendor (Phosriran and Jantama 2024). This work demonstrates the potential for large-scale valorization of DPS into high-value succinic acid.

Fig. 6.

Fig. 6

Open in a new tab

Mass balance analysis for succinic acid production from DPS through the fed-batch semi-SHF process

Conclusion

DPS is a high-volume waste material generated by the paper recycling industry. This study highlights the valorization of DPS into high-value succinic acid, a key platform chemical, via fermentation using E. coli KJ122. DPS was pre-treated with 1 N NaOH and autoclaved, followed by enzymatic hydrolysis using 60 PCU/g crude cellulase, achieving 84.6 ± 1.8% fermentable sugar release. A fed-batch semi-SHF process improved succinic acid yield and productivity up to 0.97 ± 0.01 g/g and 0.55 ± 0.02 g/L/h, respectively. Mass balance analysis indicated that 2.5 kg of NaOH-pre-treated DPS is needed per 1 kg of succinic acid produced via fermentation. This process offers a sustainable process to valorize DPS into succinic acid, supporting zero-waste and bio-circular economy principles in the paper industry.

Acknowledgements

The authors thank Hiang Seng Fiber Container Co., Ltd. (Thailand) kindly providing deinked paper sludge used in this study. The authors also thank the Office of the Permanent Secretary of the Ministry of Higher Education, Science, Research, and Innovation (Thailand) to provide financial support all activities under the university reinventing program. This work was supported by (i) Suranaree University of Technology (SUT), (ii) Thailand Science Research and Innovation (TSRI), and (iii) National Science, Research and Innovation Fund (NSRF) for a basic-research program , and for the full-time researcher fellowship program at Suranaree University of Technology (SUT) under Project No. 66/21/2567. This work also financially funded by the National Research Council of Thailand (NRCT) for a mid-career researcher development program under Grant No. NRCT5-RSA63009-04.

Author contributions

KJ conceived and designed research, and performed project administration and funding acquisition. KJ also supervised, provided methodology, investigated, curated and validated data, and finalized the final draft of manuscript. CG performed formal analysis, investigated, validated and analyzed data, and wrote the first draft of manuscript. WC helped investigating and conducting some experiments. CP and KO helped preparing and pre-treating paper sludges. KCC, KB, and CF supervised, suggested, and finalized the final draft of manuscript. All authors read and approved the manuscript.

Data availability

The research and analysis in this paper are original and truthful.

Declarations

Conflict of interest

The authors declare that they all have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

  1. Aghmashhadi OY, Asadpour G, Garmaroody ER, Zabihzadeh M, Rocha-Meneses L, Kilas T (2020) The effect of deinking process on bioethanol production from waste banknote paper. Processes 8:1563. 10.3390/pr8121563 [Google Scholar]
  2. Akhtar J, Idris A, Aziz RA (2014) Recent advances in production of succinic acid from lignocellulosic biomass. Appl Microbiol Biotechnol 98:987–1000. 10.1007/s00253-013-5319-6 [DOI] [PubMed] [Google Scholar]
  3. Bajpai P (2024) Recycling and deinking of recovered paper. Elsevier, San Diego [Google Scholar]
  4. Bayr S, Kaparaju P, Rintala J (2013) Screening pretreatment methods to enhance thermophilic anaerobic digestion of pulp and paper mill wastewater treatment secondary sludge. Chem Eng J 223:479–486. 10.1016/j.cej.2013.02.119 [Google Scholar]
  5. Borges ER, Pereira N Jr (2011) Succinic acid production from sugarcane bagasse hemicellulose hydrolysate by Actinobacillus succinogenes. J Ind Microbiol Biotechnol 38:1001–1011. 10.1007/s10295-010-0874-7 [DOI] [PubMed] [Google Scholar]
  6. Bastos JA, Remor RV, Alino JHL, Frare LM, Lofhagen JCP, Edwiges T (2021) Hydrolysate recycling improves economic feasibility of alkaline pretreatment for bioenergy production. J Environ Chem Eng 9(5):105935. 10.1016/j.jece.2021.105935 [Google Scholar]
  7. Bozell JJ, Petersen GR (2010) Technology development for the production of biobased products from biorefinery carbohydrates-the US department of energy’s “top 10” revisited. Green Chem 12:525–728. 10.1039/B922014C [Google Scholar]
  8. Budhavaram N, Fan Z (2009) Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulans strains. Bioresour Technol 100:5966–5972. 10.1016/j.biortech.2009.01.080 [DOI] [PubMed] [Google Scholar]
  9. Chan S, Jantama SS, Kanchanatawee S, Jantama K (2016) Process optimization on micro-aeration supply for high production yield of 2,3-butanediol from maltodextrin by metabolically-engineered Klebsiella oxytoca. PLoS ONE 11(9):e0161503. 10.1371/journal.pone.0161503 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen G, Song W, Qi B, Lu J, Wan Y (2013) Recycling cellulase from enzymatic hydrolysate of acid treated wheat straw by electro ultrafiltration. Bioresour Technol 144:186–193. 10.1016/j.biortech.2013.06.089 [DOI] [PubMed] [Google Scholar]
  11. Chiang CJ, HuRC HZC, Chao YP (2021) Production of succinic acid from amino acid in Escherichia coli. J Agric Food Chem 69:8172–8178. 10.1021/acs.jafc.1c02958 [DOI] [PubMed] [Google Scholar]
  12. Congthai W, Phosriran C, Chou S, Onsanoi K, Gosalawit C, Cheng KC, Jantama K (2025) Exploiting mixed waste office paper containing lignocellulosic fibers for alternatively producing high-value succinic acid by metabolically engineered Escherichia coli KJ122. Int J Mol Sci 26(3):982. 10.3390/ijms26030982 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Duncan SM, Alkasrawi M, Gurram R, Almomani F, Wiberley-Bradford AE, Singsaas E (2020) Paper mill sludge as a source of sugars for use in the production of bioethanol and isoprene. Energies 13:4662. 10.3390/en13184662 [Google Scholar]
  14. Escanciano I, Ladero M, Blanco Á, Santos VE (2024) Succinic acid production by Actinobacillus succinogenes using acid and enzymatic hydrolysates of potato and beer wastes and repeated batch operation. Biomass Bioenergy 181:107034. 10.1016/j.biombioe.2023.107034 [Google Scholar]
  15. Fennila F, Shastri Y (2016) Optimal control of enzymatic hydrolysis of lignocellulosic biomass. Resour Effic Technol 2:S96–S104. 10.1016/j.reffit.2016.11.006 [Google Scholar]
  16. Gogoi H, Nirosha V, Jayakumar A, Prabhu K, Maitra M, Panjanathan R (2018) Paper mill sludge as a renewable substrate for the production of acetone–butanol–ethanol using Clostridium sporogenes NCIM 2337. Energy Sources Part A Recover Util Environ Eff 40(1):39–44. 10.1080/15567036.2017.1405107 [Google Scholar]
  17. Gosalawit C, Kory S, Prosriran C, Jantama K (2024) Techno-economical valorization of sugarcane bagasse for efficiently producing optically pure d-(−)-lactate approaching the theoretical maximum yield in low-cost salt medium by metabolically engineered Klebsiella oxytoca. Bioresour Technol 407:131145. 10.1016/j.biortech.2024.131145 [DOI] [PubMed] [Google Scholar]
  18. Gurram RN, Al-Shannag M, Lecher NJ, Duncan SM, Singsaas EL, Alkasrawi M (2015) Bioconversion of paper mill sludge to bioethanol in the presence of accelerants or hydrogen peroxide pretreatment. Bioresour Technol 192:529–539. 10.1016/j.biortech.2015.06.010 [DOI] [PubMed] [Google Scholar]
  19. Huang M, Cheng J, Chen P, Zheng G, Wang D, Hu Y (2019) Efficient production of succinic acid in engineered Escherichia coli strains controlled by anaerobically-induced nirB promoter using sweet potato waste hydrolysate. J Environ Manag 237:147–154. 10.1016/j.jenvman.2019.02.041 [DOI] [PubMed] [Google Scholar]
  20. Jampatesh S, Sawisit A, Wong N, Jantama SS, Jantama K (2019) Evaluation of inhibitory effect and feasible utilization of dilute acid-pretreated rice straws on succinate production by metabolically engineered Escherichia coli AS1600a. Bioresour Technol 273:93–102. 10.1016/j.biortech.2018.11.002 [DOI] [PubMed] [Google Scholar]
  21. Jantama K, Zhang X, Moore JC, Shanmugam KT, Svoronos SA, Ingram LO (2008) Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C. Biotechnol Bioeng 101:881–893. 10.1002/bit.2205 [DOI] [PubMed] [Google Scholar]
  22. Johannes LP, Xuan TD (2024) Comparative analysis of acidic and alkaline pretreatment techniques for bioethanol production from perennial grasses. Energies 17:1048. 10.3390/en17051048 [Google Scholar]
  23. Jokodola EO, Narisetty V, Castro E, Durgapal S, Coulon F, Sindhu R, Binod P, Banu JR, Kumar G, Kamur V (2022) Process optimization for production and recovery of succinic acid using xylose-rich hydrolysates by Actinobacillus succinogenes. Bioresour Technol 344:126224. 10.1016/j.bioetech.2021.126224 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kádár Z, de Vrije T, BuddeMAW SZ, Réczey K, Claassen PAM (2003) Hydrogen production from paper sludge hydrolysate. Appl Biochem Biotechnol 105–108:557–566. 10.1385/ABAB:107:1-3:557 [DOI] [PubMed] [Google Scholar]
  25. Kaur R, Tyagi RD, Zhang X (2019) Review on pulp and paper activated sludge pretreatment, inhibitory effects and detoxification strategies for biovalorization. Environ Res 182:109094. 10.1016/j.envres.2019.109094 [DOI] [PubMed] [Google Scholar]
  26. Khunnonkwao P, Jantama K, Kanchanatawee S, Galier S, Roux-de Balmann H (2018) A two steps membrane process for the recovery of succinic acid from fermentation broth. Sep Purif Technol 207:451–460. 10.1016/j.seppur.2018.06.056 [Google Scholar]
  27. Khunnonkwao P, Phosriran C, In S, Kory S, Jantama K (2024) Valorization of empty oil-palm fruit bunch waste for an efficient improvement of succinic acid production by metabolically engineered Escherichia coli. Biomass Convers Biorefin 14:17149–17165. 10.1007/s13399-023-03888-5 [Google Scholar]
  28. Kim JK, Yang J, Oark SY, Yu JH, Kim KH (2019) Cellulase recycle in high-solids enzymatic hydrolysis of pretreated empty fruit bunches. Biotechnol Biofuels 12:138. 10.1186/s13068-019-1476-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lee SJ, Lee DY, Kim TY, Kim BH, Lee J, Lee SY (2005) Metabolic engineering of Escherichia coli for enhanced production of succinic acid, based on genome comparison and in silico gene knockout simulation. Appl Environ Microbiol 71:7880–7887. 10.1128/AEM.71.12.7880-7887.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lee JS, Lin CJ, Lee WC, Teng HY, Chuang MH (2022) Production of succinic acid through the fermentation of Actinobacillus succinogenes on the hydrolysate of Napier grass. Biotechnol Biofuels Bioprod 15:1–11. 10.1186/s13068-022-02106-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Loffredo R, Casalla P, Rao MA, Molino A (2024) A critical review on downstream process to recovery succinic acid from fermentation broth. Chem Eng Trans 110:169–174. 10.3303/CET24110029 [Google Scholar]
  32. Lopes AM, Filho EXF, Moreira LRS (2018) An update on enzymatic cocktail for lignocellulose breakdown. J Appl Microbiol 125:632–645. 10.1111/jam.13923 [DOI] [PubMed] [Google Scholar]
  33. Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005) Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth. Mol Cell Proteom 4(8):1205–1209 [DOI] [PubMed] [Google Scholar]
  34. Naicker JE, Govinden R, Lekha P, Sithole B (2020) Transformation of pulp and paper mill sludge (PPMS) into a glucose-rich hydrolysate using green chemistry: assessing pretreatment methods for enhanced hydrolysis. J Environ Manag 270:110914. 10.1016/j.jenvman.2020.110914 [DOI] [PubMed] [Google Scholar]
  35. Olszewska-Widdrat A, Xiros C, Wallenius A, Schneider R, de Costa PLPR, Venus J (2023) Bioprocess optimization for lactic acid and succinic acid production from pulp and paper industry side stream. Front Bioeng Biotechnol 11:1176043. 10.3389/fbioe.2023.1176043 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Orjuela A, Orjuela A, Lira CT, Miller DJ (2013) A novel process for recovery of fermentation-derived succinic acid: process design and economic analysis. Bioresour Technol 139:235–241. 10.1016/j.biorthech.2013.03.174 [DOI] [PubMed] [Google Scholar]
  37. Phosriran C, Jantama K (2024) Systematic reengineering of Klebsiella oxytoca KC004-TF160 for enhancing metabolic carbon flux towards succinate production pathway. Bioresour Technol 407:131137. 10.1016/j.biortech.2024.131137 [DOI] [PubMed] [Google Scholar]
  38. Phosriran C, Wong N, Jantama K (2024) An efficient production of bio-succinate in a novel metabolically engineered Klebsiella oxytoca by rational metabolic engineering and evolutionary adaptation. Bioresour Technol 393:130045. 10.1016/j.biortech.2023.130045 [DOI] [PubMed] [Google Scholar]
  39. Pochyba A, Samešová D, Poništ J, Salva J (2023) Pulp and paper mill sludge utilization by biological methods. Eng Proc 5:12. 10.3390/engproc2023057012 [Google Scholar]
  40. Rabemanolontsoa H, Phang ST, Kawamoto H (2024) Evaluation of various commercial enzymes for paper sludge saccharification. IOP Conf Ser Earth Environ Sci 1354(1):012020. 10.1088/1755-1315/1354/1/012020 [Google Scholar]
  41. Reyes-Sosa FM, Molales ML, Gómez AIP, Crespo NV, Zamorano LS, Rocha-Martín J, Molina-Heredia FP, García BD (2017) Management of enzyme diversity in high-performance cellulolytic cocktail. Biotechnol Biofuels Bioprod 10:1–10. 10.1186/s13068-017-0845-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Quintana E, Valls C, Roncero MB (2024) Valorization of waste paper sludge as a sustainable source for packaging applications. Polym Bull 81:9321–9345. 10.1007/s00289-024-05138-8 [Google Scholar]
  43. Sadare OO, Ejekwu O, Moshokoa MF, Jimoh MO, Daramola MO (2021) Membrane purification techniques for recovery of succinic acid obtained from fermentation broth during bioconversion of lignocellulosic biomass: current advances and future perspective. Sustainability 13(12):6794. 10.3390/su13126794 [Google Scholar]
  44. Sawisit A, Jantama SS, Kanchanatawee S, Jantama K (2015a) Efficient utilization of cassava pulp for succinate production by metabolically engineered Escherichia coli KJ122. Bioproc Biosyst Eng 38:175–187. 10.1007/s00449-014-1257-7 [DOI] [PubMed] [Google Scholar]
  45. Sawisit A, Jantama K, Zheng H, Yomano LP, York SW, Shanmugam KT, Ingram LO (2015b) Mutation in galP improved fermentation of mixed sugars to succinate using engineered Escherichiawicoli AS1600a and AM1 mineral salts medium. Bioresour Technol 193:433–441. 10.1016/j.biortech.2015.06.108 [DOI] [PubMed] [Google Scholar]
  46. Sawisit A, Jampatesh S, Jantama SS, Jantama K (2018) Optimization of sodium hydroxide pretreatment and enzyme loading for efficient hydrolysis of rice straw to improve succinate production by metabolically engineered Escherichia coli KJ122 under simultaneous saccharification and fermentation. Bioresour Technol 260:348–356. 10.1016/j.biortech.2018.03.107 [DOI] [PubMed] [Google Scholar]
  47. Show BK, Shivakumaran G, Koley A, Ghosh A, Chaudhury S, Hazra AK, Balachandran S (2023) Effect of thermal and NaOH pretreatment on water hyacinth to enhance the biogas production. Environ Sci Pollut Res 30:120984–120993. 10.1007/s11356-023-30810-3 [DOI] [PubMed] [Google Scholar]
  48. Takano M, Hoshino K (2016) Lactic acid production from paper sludge by SSF with thermotolerant Rhizopus sp. Bioresour Bioprocess 3:1–10. 10.1186/s40643-016-0106-8 [Google Scholar]
  49. Tran ATT, Cao NH, Le PTK, Mai PT, Nguyen QD (2020) Reusing alkaline solution in lignocellulose pretreatment to save consumable chemicals without losing efficiency. Chem Eng Trans 78:307–312. 10.3303/CET2078052 [Google Scholar]
  50. Umpuch C, Sakeaw S, Kanchanatawee S, Jantama K (2016) Removal of contaminated organic acids from simulated succinic acid fermentation broth by reactive extraction process: single- and mixed-solute solution. Sep Sci Technol 51:629–640. 10.1080/01496395.2015.1105823 [Google Scholar]
  51. Wang Q, Liu S, Yang G, Chen J, Ji X, Ni Y (2016) Recycling cellulose towards industrial application of enzyme treatment on hardwood kraft-based dissolving pulp. Bioresour Technol 212:160–163. 10.1016/j.biortech.2016.04.048 [DOI] [PubMed] [Google Scholar]
  52. Wang W, Wang X, Zhang Y, Yu Q, Tan X, Zhuang X, Yuan Z (2020) Effect of sodium hydroxide pretreatment on physicochemical changes and enzymatic hydrolysis of herbaceous and woody lignocelluloses. Ind Crops Prod 145:112145. 10.1016/j.indcrop.2020.112145 [Google Scholar]
  53. Wang H, Liu J, Zhang Z, Li J, Zhang H, Zhan Y (2021) Alkaline thermal pretreatment of waste activated sludge for enhanced hydrogen production in microbial electrolysis cells. J Environ Manag 294:113000. 10.1016/j.jenvman.2021.113000 [DOI] [PubMed] [Google Scholar]
  54. Werpy T, Petersen G (2004) Top value-added chemicals from biomass: Volume I—Results of screening for potential candidates from sugars and synthesis gas. 10.2172/15008859
  55. Xiao Z, Zhang X, Gregg DJ, Saddler JN (2004) Effects of sugar inhibition on cellulases and beta-glucosidase during enzymatic hydrolysis of softwood substrates. Appl Biochem Biotechnol 113:1115–1126. 10.1385/abab:115:1-3:1115 [DOI] [PubMed] [Google Scholar]
  56. Zhai R, Hu J, Saddler JN (2018) The inhibition of hemicellulosic sugars on cellulose hydrolysis are highly dependent on the cellulase productive binding, processivity, and substrate surface charges. Bioresour Technol 258:79–87. 10.1016/j.biotech.2017.12.006 [DOI] [PubMed] [Google Scholar]
  57. Zhang L, Li W, Lu J, Li R, Wu Y (2021) Production of platform and bio-fuel from paper mill sludge via hydrothermal liquefaction. J Anal Appl Pyrolysis 155:105032. 10.1016/j.jaap.2021.105032 [Google Scholar]
  58. Zhu F, Wang C, San KY, Bennett GN (2020) Metabolic engineering of Escherichia coli to produce succinate from woody hydrolysate under anaerobic conditions. J Ind Microbiol Biotechnol 47(2):223–232. 10.1007/s10295-020-02259-7 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The research and analysis in this paper are original and truthful.

Articles from 3 Biotech are provided here courtesy of Springer

RESOURCES